DRAFT
NCEA-F-0644
July 1999
Review Draft
Guidelines for
Carcinogen Risk Assessment
Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, DC
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PURPOSE OF THIS DOCUMENT
The discussion in this document is intended solely as guidance. It is not a regulation, and
does not confer legal rights or impose legal obligations on EPA, States, Tribes, local
governments, regulated entities or any member of the public.
The predominant guidance provided in this document is for EPA risk assessors to use the
best science and risk assessment techniques available to them at the time a cancer risk
assessment is conducted. Any final cancer risk assessment may take an approach different from
that described in this document based on factors such as evolving science, the facts of a
particular case, or comments from peer reviewers, the public or others.
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GUIDELINES FOR
CARCINOGEN RISK ASSESSMENT
FRL-
[To Be Developed]
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CONTENTS
LIST OF TABLES viii
LIST OF FIGURES ix
1. INTRODUCTION 1-1
1.1. PURPOSE AND SCOPE OF THE GUIDELINES 1-1
1.2. ORGANIZATION AND APPLICATION OF THE GUIDELINES 1-2
1.2.1. Organization 1-2
1.2.2. Application 1-2
1.3. USE OF DEFAULT ASSUMPTIONS 1-3
1.3.1. Default Assumptions 1-4
1.3.2. Major Defaults 1-10
1.4. CHARACTERIZATIONS 1-19
2. HAZARD ASSESSMENT 2-1
2.1. OVERVIEW OF HAZARD ASSESSMENT AND CHARACTERIZATION 2-1
2.1.1. Analyses of Data 2-1
2.1.2. Presentation of Results 2-1
2.2. ANALYSIS OF TUMOR DATA 2-2
2.2.1. Human Data 2-2
2.2.1.1. Types of Studies 2-3
2.2.1.2. Criteria for Assessing Adequacy of Epidemiologic Studies 2-4
2.2.1.3. Criteria for Causality 2-7
2.2.1.4. Assessment of Evidence of Carcinogenicity from Human Data . 2-8
2.2.2. Animal Data 2-9
2.2.2.1. Long-Term Carcinogenicity Studies 2-9
2.2.2.2. Perinatal Carcinogenicity Studies 2-15
2.2.2.3. Other Studies 2-16
2.2.3. Structural Analogue Data 2-17
2.3. ANALYSIS OF OTHER KEY DATA 2-17
2.3.1. Physicochemical Properties 2-17
2.3.2. Structure-Activity Relationships 2-18
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CONTENTS (continued)
2.3.3. Comparative Metabolism and Toxicokinetics 2-19
2.3.4. Toxicological and Clinical Findings 2-20
2.3.5. Events Relevant to Mode of Carcinogenic Action 2-21
2.3.5.1. Direct DNA Reactive Effects 2-21
2.3.5.2. Indirect DNA Effects or Other Effects on Genes/Gene
Expression 2-22
2.3.5.3. Experimental Considerations in Evaluating Data
on Precursor Events 2-24
2.3.5.4. Judging Data 2-24
2.4. BIOMARKER INFORMATION 2-25
2.5. MODE OF ACTION-GENERAL CONSIDERATIONS AND FRAMEWORK
FOR ANALYSIS 2-26
2.5.1. General Considerations 2-26
2.5.2. Evaluating a Postulated Mode of Action 2-28
2.5.3. Framework for Evaluating a Postulated Carcinogenic
Mode(s) of Action 2-29
2.5.3.1. Content of the Framework 2-30
2.6. WEIGHT-OF-EVIDENCE EVALUATION FOR POTENTIAL HUMAN
CARCINOGENICITY 2-34
2.6.1. Weight-of-Evidence Analysis 2-35
2.6.2. Descriptors for Summarizing Weight of Evidence 2-43
2.7. TECHNICAL HAZARD CHARACTERIZATION 2-45
2.8. WEIGHT-OF-EVIDENCE NARRATIVE 2-47
3. DOSE-RESPONSE ASSESSMENT 3-1
3.1. HUMAN STUDIES 3-2
3.2. MODE OF ACTION AND DOSE-RESPONSE APPROACH 3-2
3.3. DOSE-RESPONSE ANALYSIS 3-4
3.3.1. Modeling the Overall Process—Biologically Based Models 3-4
3.3.2. Analysis in the Range of Observation 3-5
3.3.2.1. Applying Information About Key Events 3-5
3.3.2.2. Procedures for Analysis in the Range of Observation of Animal
Studies 3-6
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CONTENTS (continued)
3.3.2.3. Point of Departure for Extrapolation from Observed
Animal Data 3-6
3.3.3. Analysis in the Range of Extrapolation—Default Procedures 3-9
3.3.3.1. Linear Procedure 3-9
3.3.3.2. Nonlinear Extrapolation 3-10
3.3.3.3. Linear and Nonlinear Extrapolations 3-14
3.3.3.4. Use of Toxicity Equivalence Factors and Relative Potency
Estimates 3-14
3.4. RESPONSE DATA 3-15
3.5. DOSE DATA 3-17
3.5.1. Interspecies Adjustment of Dose 3-18
3.5.2. Adjustment of Dose for Children 3-18
3.5.3. Toxicokinetic Analyses 3-20
3.5.4. Route-to-Route Extrapolation 3-21
3.5.5. Dose Averaging 3-22
3.6. DISCUSSION OF UNCERTAINTIES 3-23
3.7. TECHNICAL DOSE-RESPONSE CHARACTERIZATION 3-24
4. TECHNICAL EXPOSURE CHARACTERIZATION 4-1
5. RISK CHARACTERIZATION 5-1
5.1. PURPOSE 5-1
5.2. APPLICATION 5-2
5.3. PRESENTATION OF RISK CHARACTERIZATION SUMMARY 5-3
5.4. CONTENT OF RISK CHARACTERIZATION SUMMARY 5-3
6. REFERENCES 6-1
APPENDIX A. WEIGHT-OF-EVIDENCE NARRATIVES A-l
APPENDIX B. RESPONSES TO THE NATIONAL ACADEMY OF SCIENCES
NATIONAL RESEARCH COUNCIL REPORT SCIENCE AND JUDGMENT IN RISK
ASSESSMENT {NRC, 1994) B-l
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CONTENTS (continued)
APPENDIX C. CASE STUDY EXAMPLES FOR HAZARD EVALUATION C-l
APPENDIX D. CASE STUDY EXAMPLES FOR MODE-OF-ACTION EVALUATION . D-l
APPENDIX E. NONLINEAR DOSE-RESPONSE: MARGIN-OF-EXPOSURE
ANALYSIS (TO BE DEVELOPED) E-l
APPENDIX F. DOSE-RESPONSE ASSESSMENT FOR A CARCINOGEN POSING
HIGHER RISKS AFTER CHILDHOOD EXPOSURE F-l
APPENDIX G. RESPONSE TO COMMENTS ON OTHER SCIENCE ISSUES (TO BE
DEVELOPED) G-l
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LIST OF TABLES
Table D-l. Thyroid follicular cell tumor incidence in male rats D-2
Table D-2. Incidence of transitional cell lesions and stones
in males from a 2-year SD rat study D-l 1
Table D-3. Incidence of bladder hyperplasia and stones in male SD rats
treated up to 13 weeks D-l3
Table D-4. Reversal of incidence of bladder hyperplasia and stones following
8 weeks treatment and 16 weeks recovery D-l5
Table D-5. Clinical chemistry values (urine) in male SD rats treated up to 13 weeks . . D-17
Table D-6. Summary results of chronic bioassays D-25
Table F-l. Comparison of tumor incidence in male and female Sprague-Dawley rats
from 100 hour inhalation exposures to newborn and mature rats F-6
Table F-2 Comparison of tumor incidence in male and female Sprague-Dawley rats
From 5-wk newborn exposure and 52-wk later life exposure F-7
Table F-3 Results of PBPK modeling F-8
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1 LIST OF FIGURES
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4 Figure 1-1. Risk characterization 1-20
5 Figure 2-1. Factors for weighing human evidence 2-36
6 Figure 2-2. Factors for weighing animal evidence 2-38
7 Figure 2-3. Factors for weighing other key evidence 2-40
8 Figure 2-4. Factors for weighing totality of evidence 2-42
9 Figure 3-1. Graphical presentation of data and extrapolation 3-8
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1. INTRODUCTION
1.1. PURPOSE AND SCOPE OF THE GUIDELINES
These guidelines revise and replace United States Environmental Protection Agency
(EPA) Guidelines for Carcinogen Risk Assessment published in 51 FR 33992, September 24,
1986. The guidelines provide EPA staff and decision makers with guidance for developing and
using risk assessments. They also provide basic information to the public about the Agency's risk
assessment methods. These guidelines are used with other risk assessment guidelines that the
Agency has developed, such as the Mutagenicity Risk Assessment Guidelines (U.S. EPA, 1986c).
and the Exposure Assessment Guidelines (U.S. EPA, 1992a). Consideration of other Agency
guidance documents is particularly important when procedures for evaluating specific target organ
effects have been developed (e.g., assessment of thyroid follicular cell tumors (U.S. EPA,
1998a)), or when there is a concern for a particular sensitive subpopulation for which the Agency
has developed guidance, for example, EPA Guidelines for Developmental Toxicity Risk
Assessment (U.S. EPA, 199Id). These guidelines discuss hazards to children that may result
from exposures during preconception, prenatal, or postnatal development to sexual maturity.
Similar guidelines exist for Reproductive Toxicant Risk Assessment (U.S. EPA, 1996c) and for
Neurotoxicity Risk Assessment (U.S. EPA, 1998c). All of these guidelines should be consulted
when conducting a risk assessment in order to insure that information from studies on
carcinogenesis and other health effects are considered together in the overall characterization of
risk. This is particularly true in the case in which a precursor effect to tumor is also a precursor
or endpoint of other health effects and is used in dose-response assessment. The overall
characterization of risk will be the basis for carrying out assessments of instances in which fetuses,
infants, or children are at risk or disproportionately affected by economically significant Agency
actions. Characterization for the latter purpose is outlined in the Agency guidance by the Office
of Children's Health Protection to carry out E.O. 13045, "Protection of Children From
Environmental Health Risks and Safety Risks" issued on April 21, 1997.
The guidelines encourage both regularity in procedures to support consistency in scientific
components of Agency decision making and innovation to remain up-to-date in scientific thinking.
In balancing these goals, the Agency relies on established scientific peer review processes (EPA,
1998b). The guidelines incorporate basic principles and science policies based on evaluation of
the currently available information. As more is discovered about carcinogenesis, the need will
arise to make appropriate changes in risk assessment guidance. The Agency will revise these
guidelines when extensive changes are due. In the interim, the Agency will issue special reports,
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after appropriate peer review, to supplement and update guidance on single topics, (e.g., U.S.
EPA, 1991b). The incorporation of new, peer-reviewed scientific understanding and data in an
assessment is always consistent with the purposes of these guidelines.
1.2. ORGANIZATION AND APPLICATION OF THE GUIDELINES
1.2.1. Organization
Publications of the Office of Science and Technology Policy (OSTP, 1985) and the
National Research Council (NRC, 1983, 1994) provide information and general principles about
risk assessment. Risk assessment uses available scientific information on the properties of an
agent1 and its effects in biological systems to provide an evaluation of the potential for harm as a
consequence of environmental exposure. The 1983 and 1994 NRC documents organize risk
assessment information into four areas: hazard identification, dose-response assessment,
exposure assessment, and risk characterization. This structure appears in these guidelines, which
additionally emphasize characterization of evidence and conclusions in each part of the
assessment. In particular, the guidelines adopt the approach of the NRC's 1994 report in adding a
dimension of characterization to the hazard identification step. Added to the identification of
hazard is an evaluation of the conditions under which its expression is anticipated. The risk
assessment questions addressed in these guidelines are:
• For hazard—Can the agent present a carcinogenic hazard to humans, and if so,
under what circumstances?
• For dose-response—At what levels of exposure might effects occur?
• For exposure—What are the conditions of human exposure?
• For risk—What is the character of the risk? How well do data support conclusions
about the nature and extent of the risk?
1.2.2. Application
The guidelines apply within the framework of policies provided by applicable EPA statutes
and do not alter such policies. The guidelines cover assessment of available data. They do not
imply that one kind of data or another is prerequisite for regulatory action concerning any agent.
Risk management applies directives of regulatory legislation, which may require consideration of
potential risk, or solely hazard or exposure potential, along with social, economic, technical, and
'The term "agent" refers generally to any chemical substance, mixture, or physical or biological
entity being assessed, unless otherwise noted (See sec. 1.2.2 for a note on radiation.).
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other factors in decision making. Risk assessments support decisions, but to maintain their
integrity as decision making tools, they are not influenced by consideration of the social or
economic consequences of regulatory action.
The assessment of risk from radiation sources is based on continuing examination of
human data by the National Academy of Sciences/National Research Council in its series of
numbered reports: "Biological Effects of Ionizing Radiation". While the general principles of
these guidelines apply to radiation risk assessments, their details are most focused on other kinds
of agents. They do not attempt to guide the ongoing conduct of radiation risk assessment.
Not every EPA assessment has the same scope or depth. Agency staff often conduct
screening-level assessments for priority-setting or separate assessments of hazard or exposure for
ranking purposes or to decide whether to invest resources in collecting data for a full assessment.
Moreover, a given assessment of hazard and dose-response may be used with more than one
exposure assessment that may be conducted separately and at different times as the need arises in
studying environmental problems in various media. The guidelines apply to these various
situations in appropriate detail given the scope and depth of the particular assessment. For
example, a screening assessment may be based almost entirely on structure-activity relationships
and default assumptions. As more data become available, assessments can replace or modify
default assumptions accordingly. These guidelines do not require that all of the kinds of data
covered here be available for either assessment or decision making. The level of detail of an
assessment is a matter of Agency management discretion regarding applicable decision making
needs.
1.3. USE OF DEFAULT ASSUMPTIONS
The National Research Council, in its 1983 report on the science of risk assessment (NRC,
1983), recognized that default assumptions are necessarily made in risk assessments where gaps
exist in general knowledge or in available data for a particular agent. These default assumptions
are inferences based on general scientific knowledge of the phenomena in question and are also
matters of policy concerning the appropriate way to bridge uncertainties that concern potential
risk to human health (or, more generally, to environmental systems) from the agent under
assessment.
EPA's 1986 guidelines for cancer risk assessment (EPA, 1986b) were developed to be
responsive to the principles of the 1983 NRC report. The guidelines contained a number of
default assumptions. They also encouraged research and analysis that would lead to new risk
assessment methods and data and anticipated that these would replace defaults. The 1986
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guidelines did not explicitly discuss how to depart from defaults.
In its 1994 report on risk assessment, the NRC supported continued use of default
assumptions (NRC, 1994). The NRC report thus validated a central premise of the approach to
risk assessment that EPA had evolved in preceding years—the making of science policy inferences
to bridge gaps in knowledge—while at the same time recommending that EPA develop more
systematic and transparent guidelines to inform the public of the default inferences EPA uses in
practice. It recommended that the EPA review and update the 1986 guidelines in light of
evolving scientific information and experience in practice in applying those guidelines, and that the
EPA explain the science and policy considerations underlying current views as to the appropriate
defaults and provide general criteria to guide preparers and reviewers of risk assessments in
deciding when to depart from a default.
1.3.1. Default Assumptions
The 1994 NRC report contains several recommendations regarding flexibility and the use
of default options:
• EPA should continue to regard the use of default options as a reasonable way to
deal with uncertainty about underlying mechanisms in selecting methods and
models for use in risk assessment.
• EPA should explicitly identify each use of a default option in risk assessments.
• EPA should clearly state the scientific and policy basis for each default option.
• The Agency should consider attempting to give greater formality to its criteria for
a departure from default options in order to give greater guidance to the public and
to lessen the possibility of ad hoc, undocumented departures from default options
that would undercut the scientific credibility of the Agency's risk assessments. At
the same time, the Agency should be aware of the undesirability of having its
guidelines evolve into inflexible rules.
• EPA should continue to use the Science Advisory Board and other expert bodies.
In particular, the Agency should continue to make the greatest possible use of peer
review, workshops, and other devices to ensure broad peer and scientific
participation to guarantee that its risk assessment decisions will be based on the
best science available through a process that allows full public discussion and peer
participation by the scientific community.
In the 1983 report (p. 28), NAS defined the use of "inference options" (default options) as
a means to bridge inherent uncertainties in risk assessment. These options exist when the
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assessment encounters either "missing or ambiguous information on a particular substance" or
"gaps in current scientific theory." Since there is no instance in which a set of data on an agent or
exposure is complete, all risk assessments must use general knowledge and policy guidance to
bridge data gaps. Animal toxicity data are used, for example, to substitute for human data
because we do not test human beings. The report described the components of risk assessment in
terms of questions encountered during analysis for which inferences must be made. The report
noted (p. 36) that many components "... lack definitive scientific answers, that the degree of
scientific consensus concerning the best answer varies (some are more controversial than others),
and that the inference options available for each component differ in their degree of conservatism.
The choices encountered in risk assessment rest, to various degrees, on a mixture of scientific fact
and consensus, on informed scientific judgment, and on policy determinations (the appropriate
degree of conservatism). ..." The report did not note that the mix varies significantly from case
to case. For instance, a question that arises in hazard identification is how to use experimental
animal data when the routes of exposure differ between animals and humans. A spectrum of
inferences could be made: The most protective, or risk adverse one is that effects in animals from
one route may be seen in humans by another route. An intermediate one is a conditional inference
that such translation of effects will be assumed if the agent is absorbed by humans through the
second route. A nonprotective one that no inference is possible and the agent's effects in animals
must be tested by the second route. The choice of an inference, as the report observed, comes
from more than scientific thinking alone. While the report focused mainly on the idea of
conservatism of public health as a science policy rationale for making the choice, it did not
evaluate other considerations.
These revised guidelines retain the use of default assumptions as recommended in the
1994 report. Since the primary goal of EPA actions is public health protection and that,
accordingly, as an Agency policy, the defaults used in the absence of scientific data to the contrary
have been chosen to be health protective. The defaults described below remain public health
conservative when applied in combination in risk assessment, however, any individual default
may not constitute the most conservative position vis-a-vis that position. To do so would lead to
risk assessments that far exceed the actual risks and this would not be in keeping with the
principles discussed in the NAS 1994 report.
In addition, the guidelines reflect evaluation of experience in practice in applying defaults
and departing from them in individual risk assessments conducted under the 1986 guidelines. The
application and departure from defaults and the principles to be used in these judgments have been
matters of debate among practitioners and reviewers of risk assessments. The guidelines here are
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intended to be both explicit and more flexible than in the past concerning the basis for making
departures from defaults, recognizing that expert judgment and peer review are essential elements
of the process.
In response to the recommendations of the 1994 report, these guidelines call for
identification of the default assumptions used within assessments and for highlighting significant
issues about defaults within characterization summaries of component analyses in assessment
documents. As to the use of peer review to aid in making judgments about applying or departing
from defaults, we agree with the NRC recommendation. The Agency has long made use of
workshops, peer review of documents and guidelines, and consultations as well as formal peer
review by the Science Advisory Board (SAB). In 1998, the Administrator of EPA published a
peer review guidance for EPA scientific work products that increases the amount of peer review
for risk assessments as well as other work, continuing a series of guidance actions in response to
the NRC report and to SAB recommendations (U.S. EPA, 1994b, 1997b, 1998b).
The 1994 NRC report recommended that EPA should consider adopting principles or
criteria that would give greater formality and transparency to decisions to depart from defaults.
The report named several possible criteria for such principles (p. 7): ". . . [Protecting the public
health, ensuring scientific validity, minimizing serious errors in estimating risks, maximizing
incentives for research, creating an orderly and predictable process, and fostering openness and
trustworthiness. There might be additional relevant criteria. ..." The report indicated, however,
that the committee members had not reached consensus on a single criterion to address the key
issue of how much certainty or proof a risk assessor must have in order to justify departing from a
default. Appendix N of the report contains two presentations of alternative views held by some
committee members on this issue. One view, known as "plausible conservatism," suggested that
departures from defaults should not be made unless new information improves the understanding
of a biological process to the point that relevant experts reach consensus that the protective
default assumption concerning that process is no longer plausible. The same criterion was
recommended where the underlying scientific mechanism is well understood, but where a default
is used to address missing data. In this case, the default should not be replaced with case-specific
data unless it is the consensus of relevant experts that the proffered data make the default
assumption no longer plausible. Another view, known as the "maximum use of scientific
information" approach, acknowledged that the initial choice of defaults should be protective, but
argued that conservatism should not be a factor in determining whether to depart from the default
in favor of an alternate biological theory or alternate data. According to this view, it should not
be necessary to reach expert consensus that the default assumption had been rendered implausible;
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it should be sufficient that risk assessors find the alternate approach more plausible than the
default.
The EPA is not adopting a general list of formal decision criteria in the sense of a checklist
applicable to departures from defaults. It would not be helpful to generate a checklist of uniform
criteria. Risk assessments are highly variable in content and purpose. Screening assessments may
be purposely "worst case" in their default assumptions to eliminate problems from further
investigation. Subsequent risk assessments based on a fuller data set can discard worst-case
default assumptions in favor of plausibly protective assumptions and progressively replace or
modify the latter with data. No uniform checklist will fit all cases or all kinds of data. Moreover,
some departures from defaults are controversial, some are not. Generic checklists would likely
become more a source of rote discussion than of enlightenment about the process.
Nonetheless, for one issue, the EPA has adopted principles to give greater formality and
transparency to decisions to depart from defaults. The EPA has developed a framework for
evaluating a postulated mode of action which appears in section 2.5, below. The use of mode of
action information to make decisions about human relevance of animal data, to assist in
identifying sensitive subpopulations, and to decide upon approaches to high dose to low dose
extrapolation in dose-response assessment is a fundamental part of these guidelines. The
framework of section 2.5. contains principles derived from Bradford Hill criteria for considering
causation in human epidemiologic studies and is meant to weigh the question whether empirical
data support a mode of action that is proposed in a particular case.
The guidelines use a combination of principles and process in the application of and
departure from default assumptions. The framework of default assumptions allows risk
assessment to proceed when current scientific theory or available case-specific data do not
provide firm answers in a particular case, as the 1983 NRC report outlined. Some of the default
assumptions bridge large gaps in fundamental knowledge which will be filled by basic research on
the causes of cancer and on other biological processes, rather than by agent-specific testing.
Other default assumptions bridge smaller data gaps that can feasiblely be filled for a single agent,
such as whether a metabolic pathway in test animals is like (default) or unlike that in humans.
The decision to use a default, or not, is a choice considering available information on an
underlying scientific process and agent-specific data, depending on which kind of default it is.
Generally, if a gap in basic understanding exists, or if agent-specific data are missing, the default is
used without pause. If data are present, their evaluation may reveal inadequacies that also lead to
use of the default. If data support a plausible alternative to the default, but no more strongly than
they support the default, both the default and its alternative are carried through the assessment
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and characterized for the risk manager. If the alternative to the default are strongly supported by
data, the alternative may be used in place of the default. These guidelines provide a framework
for making such decisions. Note that, as discussed above, there is a spectrum of difficulty in
replacing default positions with empirical data. In the case of showing a mode of action, there is
need for extensive experimentation to support an hypothesis as to mode of action for a specific
tumor response, including coverage of the issue whether other modes of action are plausible.
Note that screening assessments may appropriately use "worst case" inferences to
determine if, even under those conditions, risk is low enough that a problem can be eliminated
from further consideration.
Scientific peer review, peer consultative workshops and similar processes are the principal
ways determining the strength of thinking and generally accepted views within the scientific
community about the application of and departure from defaults and about judgments concerning
the plausibility and persuasiveness of data in a particular case.
The discussion of major defaults below together with the explicit discussion of the choice
of inferences within the assessment and the processes of peer review and peer consultation (U.S.
EPA, 1998b) will serve the several goals stated in the 1994 NRC report. One is to encourage
research, since results of research efforts will be considered. Another is to allow timely decision
making, when time is a constraint, by supporting completion of the risk assessment using defaults
as needed. Another is to be flexible, using new science as it develops. Finally, the use of public
processes of peer consultation and peer review will ensure that discipline of thought is maintained
to support trust in assessment results.
There is no one set of rules for making the judgment of whether a data analysis is both
biologically plausible and persuasive as applied to the case at hand. Two criteria that apply in
these guidelines are that the underlying scientific principle has been generally accepted within the
scientific community and that supportive experiments are available that test the application of the
principle to the agent under review. For example, mutagenicity through reactivity with DNA has
been generally accepted as a carcinogenic influence for many years. This acceptance, together
with evidence of such mutagenicity in experiments on an agent, provides plausible and persuasive
support for the inference that mutagenicity is a mode of action for the agent.
Judgments about plausibility and persuasiveness of analyses vary according to the
scientific nature of the default. An analysis of data may replace a default or modify it. An
illustration of the former is development of EPA science policy on the issue of the relevance for
humans of male rat kidney neoplasia involving CC2u-globulin (U.S. EPA, 1991b). The 1991 EPA
policy gives guidance on the kind of experimental findings that demonstrate whether the
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(X2u-globulin mechanism is present and responsible for carcinogenicity in a particular case.
Before this policy guidance was issued, the default assumption was that neoplasia in question was
relevant to humans and indicated the potential for hazard to humans. A substantial body of data
was developed by public and private research groups as a foundation for the view that the a2u-
globulin induced response was not relevant to humans. These studies first addressed the a2u-
globulin mechanism in the rat and whether this mechanism has a counterpart in the human being,
both were large research efforts. The resulting data presented difficulties; some reviewers were
concerned that the mechanism in the rat appeared to be understood only in outline, not in detail,
and felt that the data were insufficient to show the lack of a counterpart mechanism in humans. It
was particularly difficult to support a negative such as the nonexistence of a mechanism in humans
because so little is known about what the mechanisms are in humans. Despite these concerns, in
its 1991 policy guidance, EPA concluded that the (X2u-globulin induced response in rats should be
regarded as not relevant to humans (i.e., as not indicating human hazard).
One conclusion from the development and peer review of this policy is that if the default
concerns an inherently complex biological question such as mode of action, large amounts of
work will be required to replace the default. A second is that "proof' in the strict sense of having
proved a negative is neither reasonable nor required. Rather the alternative may displace the
default when it is supported by clear and convincing evidence and is generally accepted in peer
review. The issue of relevance may not always be so difficult. It would be an experimentally
easier task, for example, to determine whether carcinogenesis in an animal species is due to a
metabolite of the agent in question that is not produced in humans.
When scientific processes are understood but case-specific data are missing, defaults can
be constructed to be modified by experimental data, even if data do not suffice to replace them
entirely. For example, the approaches adopted in these guidelines for scaling dose from
experimental animals to humans are constructed to be either modified or replaced as data become
available on toxicokinetic parameters for the particular agent being assessed. Similarly, the
selection of an approach or approaches for dose-response assessment is based on a series of
decisions that consider the nature and adequacy of available data in choosing among alternative
modeling and default approaches.
The 1994 NRC report notes (p. 6) that "[a]s scientific knowledge increases, the science
policy choices made by the Agency and Congress should have less impact on regulatory decision
making. Better data and increased understanding of biological mechanisms should enable risk
assessments that are less dependent on protective default assumptions and more accurate as
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predictions of human risk." Undoubtedly, this is the trend as scientific understanding increases.
However, some gaps in knowledge and data will doubtless continue to be encountered in
assessment of even data-rich cases, and it will remain necessary for risk assessments to continue
using defaults within the framework set forth here.
1.3.2. Major Defaults
This discussion covers the major default assumptions commonly employed in a cancer risk
assessment and adopted in these guidelines. They are predominantly inferences necessary to use
data observed under empirical conditions to estimate events and outcomes under environmental
conditions. Several inferential issues arise when effects seen in a subpopulation of humans or
animals are used to infer potential effects in the population of environmentally exposed humans.
Several more inferential issues arise in extrapolating the exposure-effect relationship observed
empirically to lower-exposure environmental conditions. The following issues cover the major
default areas. Typically, an issue has some sub-issues; they are introduced here, but are discussed
in greater detail in later sections.
• Is the presence or absence of effects observed in a human population predictive of
effects in another exposed human population?
• Is the presence or absence of effects observed in an animal population predictive of
effects in exposed humans?
• How do metabolic pathways relate across species? Among different age groups,
between sexes in humans?
• How do toxicokinetic processes relate across species? Among different age groups,
between sexes in humans?
• What is the correlation of the observed dose-response relationship to the relationship
at lower doses?
1.3.2.1. Is the Presence or Absence of Effects Observed in a Human Population Predictive of
Effects in Another Exposed Human Population?
When cancer effects in exposed humans are attributed to exposure to an exogenous
agent, the default assumption is that such data are predictive of cancer in any other exposed
human population. Studies either attributing cancer effects in humans to exogenous agents or
reporting no effects are often studies of occupationally exposed humans. By sex, age, and general
health, workers are not representative of the general population exposed environmentally to the
same agents. In such studies there is no opportunity to observe those who are likely to be under
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represented, e.g., fetuses, infants and children, women, or people in poor health, who may
respond differently from healthy workers. Therefore, it is understood that this assumption could
still underestimate the response of certain human subpopulations. (NRC, 1993a, 1994).
There is not enough knowledge yet to form a basis for any generally applicable, qualitative
or quantitative inference to compensate for this knowledge gap. In these guidelines, this problem
is left to analysis in individual cases, to be attended to with further general guidance as future
research and information allow. When information on a sensitive subpopulation exists, it will be
used. For example, an agent such as diethylstilbestrol (DES)causes a rare form of vaginal cancer
(clear-cell adenocarcinoma) (Herbst, 1971) in about 1 per thousand of adult women whose
mothers were exposed during pregnancy (Hatch et al., 1998). When cancer effects are not found
in an exposed human population, this information by itself is not generally sufficient to conclude
that the agent poses no carcinogenic hazard to this or other populations of potentially exposed
humans including sensitive subpopulations. This is because epidemiologic studies usually have
low power to detect and attribute responses, and typically evaluate cancer potential in a restricted
population (e.g., by age, occupation, etc.). The topic of susceptibility and variability is addressed
further in the discussion of quantitative default assumptions about dose-response relationships
below.
1.3.2.2. Is the Presence or Absence of Effects Observed in an Animal Population Predictive of
Effects in Exposed Humans?
The default assumption is that positive effects in animal cancer studies indicate that the
agent under study can have carcinogenic potential in humans. Thus, if no adequate human data
are present, positive effects in animal cancer studies are a basis for assessing the carcinogenic
hazard to humans. This assumption is a public health conservative policy, and it is both
appropriate and necessary given that we do not test for carcinogenicity in humans. The
assumption is supported by the fact that nearly all of the agents known to cause cancer in humans
are carcinogenic in animals in tests with adequate protocols (IARC, 1994; Tomatis et al., 1989;
Huff, 1994). Moreover, almost one-third of human carcinogens were identified subsequent to
animal testing (Huff, 1993). Further support is provided by research on the molecular biology of
cancer processes, which has shown that the mechanisms of control of cell growth and
differentiation are remarkably homologous among species and highly conserved in evolution.
Nevertheless, the same research tools that have enabled recognition of the nature and
commonality of cancer processes at the molecular level also have the power to reveal differences
and instances in which animal responses are not relevant to humans (Linjinsky, 1993; U.S.
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EPA, 199 lb). Under these guidelines, available mode of action2 information is studied for its
implications in both hazard and dose-response assessment and its effect on default assumptions.
There may be instances in which the use of an animal model would identify a hazard in
animals that is not truly a hazard in humans (e.g., the (X2u-globulin association with renal
neoplasia in male rats (U.S. EPA, 1991b)). The extent to which animal studies may yield false
positive indications for humans is a matter of scientific debate. To demonstrate that a response in
animals is not relevant to any human situation, adequate data to assess the relevancy issue must be
available.
The default assumption is that effects seen at the highest dose tested are appropriate for
assessment, but it is necessary that the experimental conditions be scrutinized. Animal studies
are conducted at high doses in order to provide statistical power, the highest dose being one that
is minimally toxic (maximum tolerated dose). Consequently, the question often arises whether a
carcinogenic effect at the highest dose may be a consequence of cell killing with compensatory
cell replication or of general physiological disruption, rather than inherent carcinogenicity of the
tested agent. There is little doubt that this may happen in some cases, but skepticism exists
among some scientists that it is a pervasive problem (Ames and Gold, 1990; Melnick et al., 1993a;
Melnick et al., 1993b; Barrett, 1993). If adequate data demonstrate that the effects are solely the
result of excessive toxicity rather than carcinogenicity of the tested agent per se, then the effects
may be regarded as not appropriate to include in assessment of the potential for human
carcinogenicity of the agent. This is a matter of expert judgment, considering all of the data
available about the agent including effects in other toxicity studies, structure-activity relationships,
and effects on growth control and differentiation.
When cancer effects are not found in well-conducted animal cancer studies in two or
more appropriate species and other information does not support the carcinogenic potential of
the agent, these data provide a basis for concluding that the agent is not likely to possess human
carcinogenic potential, in the absence of human data to the contrary. This default assumption
about lack of cancer effects has limitations. It is recognized that animal studies (and
epidemiologic studies as well) have very low power to detect cancer effects. Detection of a 10%
^Understanding an agent's "mode of action" means understanding the general sequence of events
by which it causes effects on cell growth control that result in cancer. "Mode of action" is used
rather than "mechanism of action" which is a term that implies complete knowledge of the steps
of carcinogenesis at the molecular level, a level of understanding that currently does not exist for
any agent.
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tumor incidence is generally the limit of power with standard protocols for animal studies (with
the exception of rare tumors that are virtually markers for a particular agent, e.g., angiosarcoma
caused by vinyl chloride). In some situations, the tested animal species may not be predictive of
effects in humans; for example, arsenic shows only minimal or no effect in animals, while it is
clearly positive in humans. Therefore, it is important to consider other information as well;
absence of mutagenic activity or absence of carcinogenic activity among structural analogues, can
increase the confidence that negative results in animal studies indicate a lack of human hazard.
Another limitation is that standard animal study protocols are not yet available for effectively
studying perinatal effects. The potential for effects on the very young generally must be
considered separately. Perinatal studies accomplished by modification of existing adult bioassay
protocols need to be required in special circumstances under existing Agency policy (U.S. EPA,
1997a,b)
The default assumption is that target organ concordance is not a prerequisite for
evaluating the implications of animal study results for humans. Target organs of carcinogenesis
for agents that cause cancer in both animals and humans are most often concordant at one or
more sites (Tomatis et al., 1989; Huff, 1994). However, concordance by site is not uniform.
The mechanisms of control of cell growth and differentiation are concordant among species, but
there are marked differences among species in the way control is managed in various tissues. For
example, in humans, mutations of the tumor suppressor genes p53 and retinoblastoma are
frequently observed genetic changes in tumors. These tumor suppressor genes are also observed
to be operating in some rodent tissues, but other growth control mechanisms predominate in other
rodent tissues. Thus, an animal response may be due to changes in a control that are relevant to
humans, but appear in animals in a different way. However, it is appropriate under these
guidelines to consider the influences of route of exposure, metabolism, and, particularly, some
modes of action that may either support or not support target organ concordance between animals
and humans. When data allow, these influences are considered in deciding whether the default
remains appropriate in individual instances (NRC, 1994, p. 121). Another exception to the basic
default of not assuming site concordance exists in the context of toxicokinetic modeling. Site
concordance is inherently assumed when these models are used to estimate delivered dose in
humans based on animal data.
The default is to include benign tumors observed in animal studies in the assessment of
animal tumor incidence if they have the capacity to progress to the malignancies with which they
are associated. This default is consistent with the approach of the National Toxicology Program
and the International Agency for Research on Cancer and is somewhat more protective of public
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health than not including benign tumors in the assessment. This treats the benign and malignant
tumors as representative of related responses to the test agent (McConnell et al., 1986), which is
scientifically appropriate. Nonetheless, in assessing findings from animal studies, a greater
proportion of malignancy is weighed more heavily than a response with a greater proportion of
benign tumors. Greater frequency of malignancy of a particular tumor type in comparison with
other tumor responses observed in an animal study is also a factor to be considered in selecting
the response to be used in dose-response assessment.
Benign tumors that are not observed to progress to malignancy are assessed on a case-
by-case basis. There is a range of possibilities for their overall significance. They may deserve
attention because they are serious health problems even though they are not malignant; for
instance, benign tumors may be a health risk because of their effect on the function of a target
tissue such as the brain. They may be significant indicators of the need for further testing of an
agent if they are observed in a short term test protocol, or such an observation may add to the
overall weight of evidence if the same agent causes malignancies in a long term study.
Knowledge of the mode of action associated with a benign tumor response may aid in the
interpretation of other tumor responses associated with the same agent.
1.3.2.3. How Do Metabolic Pathways Relate Across Species? Among different age groups,
between sexes in humans?
The default assumption is that there is a similarity of the basic pathways of metabolism
and the occurrence of metabolites in tissues in regard to the species-to-species extrapolation of
cancer hazard and risk. If comparative metabolism studies were to show no similarity between
the tested species and humans and a metabolite(s) were the active form, there would be less
support for an inference that the animal response(s) relates to humans. In other cases, parameters
of metabolism may vary quantitatively between species; this becomes part of deciding on an
appropriate human equivalent dose based on animal studies, optimally in the context of a
toxicokinetic model. While the basic pathways are assumed to be the same among humans, the
presence of polymorphisms and the maturation of the pathways in infants needs to be considered.
The active form of an agent may be present to differing degrees, or completely absent, which may
result in greater or lesser risk for subpopulations.
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1.3.2.4. How Do Toxicokinetic Processes Relate Across Species? Among different age
groups, between sexes in humans?
A major issue is how to estimate human equivalent doses in extrapolating from animal
studies. As a default for oral exposure, a human equivalent dose for adults is estimatedfrom
data on another species by an adjustment of animal applied oral dose by a scaling factor of body
weight to the 0.75 power. This adjustment factor is used because it represents scaling of metabolic
rate across animals of different size. Because the factor adjusts for a parameter that can be
improved on and brought into more sophisticated toxicokinetic modeling, when such data become
available, the default assumption of 0.75 power can be refined or replaced. The same factor is
usedfor children because it is slightly more protective than using children's body weight (see
section 1.3.5.2).
For inhalation exposure, a human equivalent dose for adults is estimated by default
methodologies that provide estimates of lung deposition and of internal dose. The methodologies
can be refined to more sophisticated forms with data on toxicokinetic and metabolic parameters of
the specific agent. This default assumption, like the one with oral exposure, is selected in part
because it lays a foundation for incorporating better data. Because of the differences for infants
and children, for gases and aerosols, an adjustment is made for their breathing rate and their
body weight. For inhaled particles, the adjustment does not take into account the different size
and spacing of airways of children and adults; this difference could result in children and adults
retaining particles with a different size distribution and different toxicologic properties. To reduce
this uncertainty, EPA is developing a default dosimetry model for children that is based on
children's inhalation parameters. The use of information to improve dose estimation from applied
to internal to delivered dose is encouraged, including use of toxicokinetic modeling instead of any
default, where data are available.
The processes of absorption, distribution, and elimination have important differences
among infants, adults, and older adults, e.g., infants tend to absorb metals through the gut more
rapidly and more efficiently than older children or adults (Calabrese, 1986). Renal elimination is
also not as efficient in infants. While these processes reach adult compentency at about the time
of weaning, they may have important implications, particularly when the dose-response
relationship for an agent is considered to be nonlinear and there is an exposure scenario
disproportionately affecting infants, because in these cases the magnitude of dose is more
pertinent than the usual approach in linear extrpolation, of averaging dose across a lifetime.
Efficiency of intestinal absorption in older adults tends to be generally less overall for most
chemicals. Another notable difference is that, post-weaning (about one year), children have a
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higher metabolic rate than adults (Renwick, 1999) and may toxify or detoxify agents at a
correspondingly higher rate..
For a route-to-route of exposure extrapolation, the default assumption is that an agent
that causes internal tumors by one route of exposure will be carcinogenic by another route if it is
absorbed by the second route to give an internal dose. This is a qualitative assumption and is
considered to be public health conservative. The rationale is that for internal tumors an internal
dose is significant no matter what the route of exposure. Additionally, the metabolism of the
agent will be qualitatively the same for an internal dose. The issue of quantitative extrapolation of
the dose-response relationship from one route to another is addressed case by case. Quantitative
extrapolation is complicated by considerations such as first-pass metabolism, but is approachable
with empirical data. Adequate data are necessary to demonstrate that an agent will act differently
by one route versus another route of exposure.
1.3.2.5. What Is the Correlation of the Observed Dose-Response Relationship to the
Relationship at Lower Doses?
If sufficient data are available, a biologically based model for both the observed range and
extrapolation below that range may be used. While no standard biologically based models are in
existence, one may be developed if extensive data exist in a particular case and the purpose of the
assessment justifies the investment of resources needed. The default procedure for the observed
range of data, when a biologically based model is not used, is to use a curve-fitting model for
incidence data.
In the absence of data supporting a biologically based model for extrapolation outside of
the observed range, the choice of approach is based on the view of mode of action of the agent
arrived at in the hazard assessment.
The basic default is to assume linearity and use a linear default approach when the mode
of action information is supportive of linearity or mode of action is not understood. The linear
approach is used when a view of the mode of action indicates a linear response, for example,
when a conclusion is made that an agent directly causes alterations in DNA, a kind of interaction
that not only theoretically requires one reaction, but also is likely to be additive to ongoing,
spontaneous gene mutation. Other kinds of activity may have linear implications, e.g., linear rate-
limiting steps, that support a linear procedure also. The linear approach is to draw a straight line
between a point of departure from observed data, generally, as a default, the LED10, and the
origin (zero incremental dose, zero incremental response). Other points of departure may be
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more appropriate for certain data sets; these may be used instead of the LED10. This approach is
generally considered to be public health protective. The LED10 is the lower 95% limit on a dose
that is estimated to cause a 10% response. This level is chosen to account (protectively) for
experimental variability. Additionally, it is chosen because it rewards experiments with better
designs in regard to number of doses and dose spacing, since these generally will have narrower
confidence limits. It is also an appropriate representative of the lower end of the observed range
because the limit of detection of studies of tumor effect is about 10%.
The linear default is thought to generally provide an upper bound calculation of potential
risk at low doses, e.g., a 1/100,000 to 1/1,000,000 risk; the straight line approach gives numerical
results about the same as a linearized multistage procedure. This upper bound is thought to be
public health conservative at low doses for the range of human variability considering the typical
Agency target range for risk management of 1/1,000,000 to 1/10,000, although it may not
completely do so (Bois et al., 1995) if pre-existing disease or genetic constitution place a
percentage of the population at risk from any exposure above zero to xenobiotics, natural or
manmade. The question of what may be the actual variability in human sensitivity is one that the
1994 NRC report discussed as did the 1993 NRC report on pesticides in children and infants. The
NRC has recommended research on the question, and the EPA and other agencies are conducting
such research. Given the current state of knowledge, the EPA will assume that the linear default
procedure adequately accounts for human variability unless there is case-specific information for a
given agent that indicates a particularly sensitive subpopulation, in which case the special
information will be used.
When adequate data on mode of action show that linearity is not plausible, and provide
sufficient evidence to support a nonlinear mode of action for the general population and any
subpopulations of concern, the default changes to a different approach— a margin of exposure
analysis—which assumes that nonlinearity is more reasonable. The departure point is again
generally the LED10 when incidence data are modeled. When the data available are continuous
data such as blood levels of hormones or organ weight, a NOAEL/LOAEL procedure is typically
used since modeling approaches for deriving a point of departure from continuous data are not yet
available. Until these modeling approaches are developed and adopted, continuous data and data
sets that are a mixture of incidence and continuous data can be examined by the NOAEL/LOAEL
procedure. In the nonlinear approach, the margin that exists between a human exposure of interest
and the point of departure is examined for adequacy to protect public health. A margin of
exposure analysis may be used as the basis to consider the protectiveness of a possible
environmental criterion for regulation or to judge whether an existing exposure might present risk.
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A sufficient basis to support this nonlinear procedure will include data on responses that
are key events3 integral to the carcinogenic process. This means that the point of departure
mostly will be from these precursor response data, e.g., hormone levels, mitogenic effects, rather
than tumor incidence data.
The mode of action may have specific implications to be considered for risk potential of
certain exposure scenarios. For instance, stimulus of cell growth through hormonal or other signal
disruption or as a result of damage from toxicity are reversible if the exposure is for a short time
since homeostasis brings a return to normal levels after cessation of exposure. Another feature of
a specific exposure scenario may be the exposure of a sensitive subpopulation. If the population
exposed in a particular scenario is wholly or largely composed of a subpopulation for whom
evidence indicates a special sensitivity to the agent's mode of action, an adequate margin of
exposure would be larger than for general population exposure.
When the mode of action information indicates that the dose-response may be adequately
described by both a linear and a nonlinear approach, then the default is to present both the
linear and margin of exposure analyses. An assessment may use both linear and nonlinear
approaches if linearity is not plausible and nonlinearity has support, but a mode of action is not
defined, or different responses are thought to result from different modes of action or a response
appears to be very different at high and low doses due to influence of separate modes of action.
The results may be needed for assessment of combined risk from agents with common modes of
action.
A default assumption is made that cumulative dose received over a lifetime, expressed as
a lifetime average daily dose, is an appropriate measure of dose. This assumes that a high dose
of such an agent received over a shorter period of time is equivalent to a low dose spread over a
lifetime. This is thought to be a relatively public health protective assumption and has empirical
support (Monro, 1992). An example of effects of short-term, high exposure that results in
subsequent cancer development is treatment of cancer patients with certain chemotherapeutic
agents. An example of cancer from long-term exposure to an agent of relatively low potency is
smoking. When sufficient information is available indicating that the carcinogenic mode of action
supports a nonlinear dose-response approach, a different approach may be used. Such an
approach includes considering the margin of exposure that exists between exposure and the point
of departure from the observed data range. In these cases, short-term exposure estimates (several
days to several months may be more appropriate than the lifetime average daily dose. In these
3A "key event" is an empirically observed precursor consistent with a mode of action.
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cases both agent concentration and duration are likely to be important, because such effects are
generally observed to be reversible at cessation of very short-term exposure.
1.4. CHARACTERIZATIONS
The risk characterization process first summarizes findings on hazard, dose-response, and
exposure characterizations, then develops an integrative analysis of the whole risk case. It ends in
a non technical Risk Characterization Summary. The Risk Characterization Summary is a
presentation for risk managers who may or may not be familiar with the scientific details of cancer
assessment. It also provides information for other interested readers. The initial steps in the risk
characterization process are to make building blocks in the form of characterizations of the
assessments of hazard, dose-response, and exposure. The individual assessments and
characterizations are then integrated to arrive at risk estimates for exposure scenarios of interest.
As part of the characterization process, explicit evaluations will be made of the hazard and risk
potential for susceptible populations, including children (U.S EPA 1995a,b). There are two
reasons for individually characterizing the hazard, dose-response, and exposure assessments. One
is that they are often done by different people than those who do the integrative analyses. The
second is that there is very often a lapse of time between the conduct of hazard and dose-
response analyses and the conduct of exposure assessment and integrative analysis. Thus, it is
necessary to capture characterizations of assessments as the assessments are done to avoid the
need to go back and reconstruct them. Finally, frequently a single hazard assessment is used by
several programs for several different exposure scenarios. Figure 1-2 shows the relationships of
analyses. The figure does not necessarily correspond to the number of documents involved; there
may be one or several. "Integrative analysis" is a generic term. At EPA, the documents of
various programs that contain integrative analyses have other names such as the "Staff Paper" that
discusses air quality criteria issues. In the following sections, the elements of this figure are
discussed.
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CHARACTERIZATIONS
T echmcal
Hazard
Charaeterliaion
Technical
Dom Rttpontt
Characterization
Technical
Exposure
Characterization
Hazard
Assessment
Dose Response
Assessment
RISK
CHARACTERIZATION
SUMMARY
Risk Characterization Process
Figure 1-1. Risk Characterization
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2. HAZARD ASSESSMENT
2.1. OVERVIEW OF HAZARD ASSESSMENT AND CHARACTERIZATION
2.1.1. Analyses of Data
The purpose of hazard assessment is to review and evaluate data pertinent to two
questions: (1) whether an agent may pose a carcinogenic hazard to human beings and (2) under
what circumstances an identified hazard may be expressed (NRC, 1994, p. 142). Hazard
assessment is composed of analyses of a variety of data that may range from observations of
tumor responses to analysis of structure-activity relationships. The purpose of the assessment is
not simply to assemble these separate evaluations; its purpose is to construct a total case analysis
examining the biological story the data reveal as a whole about carcinogenic effects, mode of
action, and implications of these for human hazard and dose-response evaluation. Weight-of-
evidence conclusions come from the combined strength and coherence of inferences appropriately
drawn from all of the available evidence. To the extent that data permit, hazard assessment
addresses the question of mode of action as both an initial step in identifying human hazard
potential and as a part of considering appropriate approaches to dose-response assessment.
The topics in this chapter include analysis of tumor data, both animal and human, and
analysis of other key information about properties and effects that relate to carcinogenic potential.
The chapter addresses how information can be used to evaluate potential modes of action. It also
provides guidance on performing a weight-of-evidence evaluation.4
2.1.2. Presentation of Results
Presentation of the results of hazard assessment follows Agency guidance as discussed in
Section 2.7. The results are presented in a technical hazard characterization that serves as a
support to later risk characterization. It includes:
• a summary of the evaluations of hazard data,
• the rationales for its conclusions, and
• an explanation of the significant strengths or limitations of the conclusions.
Another presentation feature is the use of a weight-of-evidence narrative that includes
'"Mode" of action is contrasted with "mechanism" of action, which implies a more detailed,
molecular description of key processes and events than is meant by mode of action.
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both a conclusion about the weight-of-evidence of carcinogenic potential and a summary of the
data on which the conclusion rests. This narrative is a brief summary that replaces the
alphanumerical classification system used in EPA's previous guidelines.
2.2. ANALYSIS OF TUMOR DATA
Evidence of carcinogenicity comes from finding tumor increases in humans or laboratory
animals exposed to a given agent, or from finding tumors following exposure to structural
analogues to the compound under review. The significance of observed or anticipated tumor
effects is evaluated in reference to all the other key data on the agent. This section contains
guidance for analyzing human and animal studies to decide whether there is an association
between exposure to an agent or a structural analogue and occurrence of tumors. Note that the
use of the term "tumor" here is generic, meaning malignant neoplasms or a combination of
malignant and corresponding benign neoplasms.
Observation of only benign neoplasia may or may not have significance. Benign tumors
that are not observed to progress to malignancy are assessed on a case-by-case basis. There is a
range of possibilities for their overall significance. They may deserve attention because they are
serious health problems even though they are not malignant; for instance, benign tumors may be a
health risk because of their effect on the function of a target tissue such as the brain. They may be
significant indicators of the need for further testing of an agent if they are observed in a short-
term test protocol, or such an observation may add to the overall weight of evidence if the same
agent causes malignancies in a long-term study. Knowledge of the mode of action associated with
a benign tumor response may aid in the interpretation of other tumor responses associated with
the same agent. In other cases, observation of a benign tumor response alone may have no
significant health hazard implications when other sources of evidence show no suggestion of
carcinogenicity.
2.2.1. Human Data
Human data may come from epidemiologic studies or case reports. Epidemiology is the
study of the distributions and causes of disease within human populations. The goals of cancer
epidemiology are to identify differences in cancer risk between different groups in a population or
between different populations, and then to determine the extent to which these differences in risk
can be attributed causally to specific exposures to exogenous or endogenous factors.
Epidemiologic data are extremely useful in risk assessment because they provide direct evidence
that a substance produces cancer in humans, thereby avoiding the problem of species-to-species
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inference. Thus, when available human data are extensive and of good quality, they are generally
preferable over animal data and should be given greater weight in hazard characterization and
dose-response assessment, although both are utilized.
Null results from a single epidemiologic study cannot prove the absence of carcinogenic
effects because they can arise either from being truly negative or from inadequate statistical
power, inadequate design, imprecise estimates, or confounding factors. However, null results
from a well-designed and well-conducted epidemiologic study that contains usable exposure data
can help to define upper limits for the estimated dose of concern for human exposure if the overall
weight of the evidence indicates that the agent is potentially carcinogenic in humans.
Epidemiology can also complement experimental evidence in corroborating or clarifying
the carcinogenic potential of the agent in question. For example, observations from epidemiologic
studies that elevated cancer incidence occurs at sites corresponding to those at which laboratory
animals experience increased tumor incidence can strengthen the weight of evidence of human
carcinogenicity. On the other hand, strong nonpositive epidemiologic data alone or in conjunction
with compelling mechanistic information can lend support to a conclusion that animal responses
may not be predictive of a human response. Furthermore, the advent of biochemical or molecular
epidemiology may help improve understanding of the mechanisms of human carcinogenesis.
2.2.1.1. Types of Studies
The major types of cancer epidemiologic studies are analytical studies and descriptive or
correlation studies. Each study type has well-known strengths and weaknesses that affect
interpretation of results as summarized below (Kelsey et al., 1986; Lilienfeld and Lilienfeld, 1979;
Mausner and Kramer, 1985; Rothman, 1986).
Analytical epidemiologic studies are most useful for identifying an association between
human exposure and adverse health effects. Analytical study designs include case-control studies
and cohort studies. In case-control studies, groups of individuals with (cases) and without
(controls) a particular disease are identified and compared to determine differences in exposure.
In cohort studies, a group of "exposed" and "nonexposed" individuals are identified and studied
over time to determine differences in disease occurrence. Cohort studies can either be performed
prospectively, or retrospectively from historical records.
Descriptive or correlation epidemiologic studies (sometimes called ecological studies)
examine differences in disease rates among populations in relation to age, gender, race, and
differences in temporal or environmental conditions. In general, these studies can only identify
patterns or trends in disease occurrence over time or in different geographical locations, but
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cannot ascertain the causal agent or degree of exposure. These studies, however, are often very
useful for generating hypotheses for further research.
Biochemical or molecular epidemiologic studies are studies in which laboratory methods
are incorporated in analytical investigations. The application of techniques for measuring cellular
and molecular alterations due to exposure to specific environmental agents may allow conclusions
to be drawn about the mechanisms of carcinogenesis. The use of biological biomarkers in
epidemiology may improve assessment of exposure and internal dose.
Case reports describe a particular effect in an individual or group of individuals who were
exposed to a substance. These reports are often anecdotal or highly selected in nature and are of
limited use for hazard assessment. However, reports of cancer cases can identify associations,
particularly when there are unique features such as an association with an uncommon tumor (e.g.,
vinyl chloride and angiosarcoma or diethylstilbestrol and clear-cell carcinoma of the vagina).
2.2.1.2. Criteria for Assessing Adequacy of Epidemiologic Studies
Criteria for assessing the adequacy of epidemiologic studies are well recognized.
Characteristics that are desirable in these studies include (1) clear articulation of study objectives
or hypothesis; (2) proper selection and characterization of the exposed and control groups; (3)
adequate characterization of exposure; (4) sufficient length of follow-up for disease occurrence;
(5) valid ascertainment of the causes of cancer morbidity and mortality; (6) proper consideration
of bias and confounding factors; (7) adequate sample size to detect an effect; (8) clear, well-
documented, and appropriate methodology for data collection and analysis; (9) adequate response
rate and methodology for handling missing data; and (10) complete and clear documentation of
results. Ideally, these conditions should be satisfied, where appropriate, but rarely can a study
meet all of them. No single criterion determines the overall adequacy of a study. The following
discussions highlight the major factors included in an analysis of epidemiologic studies.
Population Issues
The ideal comparison would be between two populations that differ only in exposure to
the agent in question. Because this is seldom the case, it is important to identify sources of bias
inherent in a study's design or data collection methods. Bias can arise from several sources,
including noncomparability between populations of factors such as general health (McMichael,
1976), diet, lifestyle, or geographic location; differences in the way case and control individuals
recall past events; differences in data collection that result in unequal ascertainment of health
effects in the populations; and unequal follow-up of individuals. Both acceptance of studies for
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assessment and judgment of their strengths or weaknesses depend on identifying their sources of
bias and the effects on study results.
Exposure Issues
For epidemiologic data to be useful in determining whether there is an association between
health effects and exposure to an agent, there must be adequate characterization of exposure
information. In general, greater weight should be given to studies with more precise and specific
exposure estimates.
Questions to address about exposure are: What can one reliably conclude about the level,
duration, route, and frequency of exposure of individuals in one population as compared with
another? How sensitive are study results to uncertainties in these parameters?
Actual exposure measurements are not available for many retrospective studies.
Therefore, surrogates are often used to reconstruct exposure parameters. These may involve
attributing exposures to job classifications in a workplace or to broader occupational or
geographic groupings. Use of surrogates carries a potential for misclassification in that
individuals may be placed in an incorrect exposure group. Misclassification generally leads to
reduced ability of a study to detect differences between study and referent populations.
When either current or historical monitoring data are available, the exposure evaluation
includes consideration of the error bounds of the monitoring and analytic methods and whether
the data are from routine or accidental exposures. The potentials for misclassification and
measurement errors are amenable to both qualitative and quantitative analysis. These are essential
analyses forjudging a study's results because exposure estimation is the most critical part of a
retrospective study.
Biological markers potentially offer excellent measures of exposure (Hulka and Margolin,
1992; Peto and Darby, 1994). Validated markers of exposure such as alkylated hemoglobin from
exposure to ethylene oxide (van Sittert et al., 1985) or urinary arsenic (Enterline et al., 1987) can
greatly improve estimates of dose. Markers closely identified with effects promise to greatly
increase the ability of studies to distinguish real effects from bias at low levels of relative risk
between populations (Taylor et al., 1994; Biggs et al., 1993) and to resolve problems of
confounding risk factors.
Confounding Factors
Because epidemiologic studies are mostly observational, it is not possible to guarantee the
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control of confounding variables, which may affect the study outcome. A confounding variable is
a risk factor, independent of the putative agent, that is distributed unequally among the exposed
and unexposed populations (e.g., smoking habits, lifestyle). Adjustment for possible confounding
factors can occur either in the design of the study (e.g., matching on critical factors) or in the
statistical analysis of the results. The influence of a potential confounding factor is limited by the
effect of the exposure of interest. For example, a twofold effect of an exposure requires that the
confounder effect be at least as big. The latter may not be possible owing to the presentation of
the data or because needed information was not collected during the study. In this case, indirect
comparisons may be possible. For example, in the absence of data on smoking status among
individuals in the study population, an examination of the possible contribution of cigarette
smoking to increased lung cancer risk may be based on information from other sources such as
the American Cancer Society's longitudinal studies (Hammand, 1966; Garfinkel and Silverberg,
1991). The effectiveness of adjustments contributes to the ability to draw inferences from a
study.
Different studies involving exposure to an agent may have different confounding factors.
If consistent increases in cancer risk are observed across a collection of studies with different
confounding factors, the inference that the agent under investigation was the etiologic factor is
strengthened, even though complete adjustment for confounding factors cannot be made and no
single study supports a strong inference.
It also may be the case that the agent of interest is a risk factor in conjunction with another
agent. This relationship may be revealed in a collection of studies such as in the case of asbestos
exposure and smoking.
Sensitivity
Sensitivity, or the ability of a study to detect real effects, is a function of several factors.
Greater size of the study population(s) (sample size) increases sensitivity, as does greater
exposure (levels and duration) of the population members. Because of the often long latency
period in cancer development, sensitivity also depends on whether adequate time has elapsed
since exposure began for effects to occur. A unique feature that can be ascribed to the effects of
a particular agent (such as a tumor type that is seen only rarely in the absence of the agent) can
increase sensitivity by permitting separation of bias and confounding factors from real effects.
Similarly, a biomarker particular to the agent can permit these distinctions. Statistical re-analyses
of data, particularly an examination of different exposure indices, can give insight on potential
exposure-response relationships. These are all factors to explore in statistical analysis of the data.
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Statistical Considerations
The analysis applies appropriate statistical methods to ascertain whether or not there is
any significant association between exposure and effects. A description of the method or methods
should include the reasons for their selection. Statistical analyses of the potential effects of bias or
confounding factors are part of addressing the significance of an association, or lack of one, and
whether a study is able to detect any effect.
The analysis augments examination of the results for the whole population with
exploration of the results for groups with comparatively greater exposure or time since first
exposure. This may support identifying an association or establishing a dose-response trend.
When studies show no association, such exploration may apply to determining an upper limit on
potential human risk for consideration alongside results of animal tumor effects studies.
Combining Statistical Evidence Across Studies
Meta-analysis is a means of comparing and synthesizing studies dealing with similar health
effects and risk factors. It is intended to introduce consistency and comprehensiveness into what
otherwise might be a more subjective review of the literature. When utilized appropriately, meta-
analysis can enhance understanding of associations between sources and their effects that may not
be apparent from examination of epidemiologic studies individually. Whether to conduct a meta-
analysis depends on several issues. These include the importance of formally examining sources
of heterogeneity, the refinement of the estimate of the magnitude of an effect, and the need for
information beyond that provided by individual studies or a narrative review. Meta-analysis may
not be useful in some circumstances. These include when the relationship between exposure and
disease is obvious without a more formal analysis; when there are only a few studies of the key
health outcomes; when there is insufficient information from available studies related to disease,
risk estimate, or exposure classification; or when there are substantial confounding or other biases
that cannot be adjusted for in the analysis (Blair et al., 1995; Greenland, 1987; Peto, 1992).
2.2.1.3. Criteria for Causality
A causal interpretation is enhanced for studies to the extent that they meet the criteria
described below. None of the criteria is conclusive by itself, and the only criterion that is essential
is the temporal relationship. These criteria are modeled after those developed by Bradford Hill in
the examination of cigarette smoking and lung cancer (Rothman, 1986), and they need to be
interpreted in the light of all other information on the agent being assessed.
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Temporal relationship: The development of cancers requires certain latency
periods, and while latency periods vary, existence of such periods is generally
acknowledged. Thus, the disease has to occur within a biologically reasonable
time after initial exposure. This feature must be present if causality is to be
considered.
Consistency: Associations occur in several independent studies of a similar
exposure in different populations, or associations occur consistently for different
subgroups in the same study. This feature usually constitutes strong evidence for a
causal interpretation when the same bias or confounding is not also duplicated
across studies.
Magnitude of the association: A causal relationship is more credible when the risk
estimate is large and precise (narrow confidence intervals).
Biological gradient: The risk ratio (i.e., the ratio of the risk of disease or death
among the exposed to the risk of the unexposed) increases with increasing
exposure or dose. Statistical significance is important, and a strong dose-response
relationship across several categories of exposure, latency, and duration is
supportive for causality, given that confounding is unlikely to be correlated with
exposure. The absence of a dose-response relationship, however, is not by itself
evidence against a causal relationship.
Specificity of the association: The likelihood of a causal interpretation is increased
if an exposure produces a specific effect (one or more tumor types also found in
other studies) or if a given effect has a unique exposure.
Biological plausibility: The association makes sense in terms of biological
knowledge. Information is considered from animal toxicology, toxicokinetics,
structure-activity relationship analysis, and short-term studies of the agent's
influence on events in the carcinogenic process considered.
Coherence: The cause-and-effect interpretation is in logical agreement with what
is known about the natural history and biology of the disease, i.e., the entire body
of knowledge about the agent.
2.2.1.4. Assessment of Evidence of Carcinogenicity from Human Data
In the evaluation of carcinogenicity based on epidemiologic studies, it is necessary to
critically evaluate each study for confidence in findings and conclusions as discussed under
Section 2.2.1.2. All studies that are properly conducted, whether yielding positive or null results,
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or even suggesting protective carcinogenic effects, should be considered in assessing the totality
of the human evidence. Although a single study may be indicative of a cause-effect relationship,
confidence in inferring a causal relationship is increased when several independent studies are
concordant in showing the association, when the association is strong, and when other criteria for
causality are also met. Conclusions about the overall evidence for carcinogenicity from available
studies in humans should be summarized along with a discussion of strengths or limitations of the
conclusions.
2.2.2. Animal Data
Various whole-animal test systems are currently used or are under development for
evaluating potential carcinogenicity. Cancer studies involving chronic exposure for most of the
lifespan of an animal are generally accepted for evaluation of tumor effects (Tomatis et al., 1989;
Rail, 1991; Allen et al., 1988; but see Ames and Gold, 1990). Other studies of special design are
useful for observing formation of preneoplastic lesions or tumors or investigating specific modes
of action. Their applicability is made on a case-by-case basis.
2.2.2.1. Long-Term Carcinogenicity Studies
The objective of long-term carcinogenesis bioassays is to determine the potential
carcinogenic hazard and dose-response relationships of the test agent. Carcinogenicity rodent
studies are designed to examine the production of tumors as well as preneoplastic lesions and
other indications of chronic toxicity that may provide evidence of treatment-related effects and
insights into the way the test agent produces tumors. Current standardized carcinogenicity
studies in rodents test at least 50 animals per sex per dose group in each of three treatment groups
and in a concurrent control group, usually for 18 to 24 months, depending on the rodent species
tested (OECD, 1981; U.S. EPA, 1983a-c). The high dose in long-term studies is generally
selected to provide the maximum ability to detect treatment-related carcinogenic effects while not
compromising the outcome of the study through excessive toxicity or inducing inappropriate
toxicokinetics (e.g., overwhelming absorption or detoxification mechanisms). The purpose of two
or more lower doses is to provide some information on the shape of the dose-response curve.
Similar protocols have been and continue to be used by many laboratories worldwide.
All available studies of tumor effects in whole animals are considered, at least
preliminarily. The analysis discards studies judged to be wholly inadequate in protocol, conduct,
or results. Criteria for the technical adequacy of animal carcinogenicity studies have been
published and should be used as guidance to judge the acceptability of individual studies (NTP,
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1984; OSTP, 1985). Care is taken to include studies that provide some evidence bearing on
carcinogenicity or that help interpret effects noted in other studies, even if they have some
limitations of protocol or conduct. Such limited, but not wholly inadequate, studies can
contribute as their deficiencies permit. The findings of long-term rodent bioassays are always
interpreted in conjunction with results of prechronic studies along with metabolism toxicokinetic
metabolism studies and other pertinent information, if available. Evaluation of tumor effects
requires consideration of both biological and statistical significance of the findings (Haseman,
1984, 1985, 1990, 1995). The following sections highlight the major issues in the evaluation of
long-term carcinogenicity studies.
Dosing Issues
Among the many criteria for technical adequacy of animal carcinogenicity studies is the
appropriateness of dose selection. The selection of doses for chronic bioassays requires scientific
judgments and must be based on sound toxicologic principles. Dose selection should be made on
the basis of relevant toxicologic information from prechronic, mechanistic, and toxicokinetic and
mechanistic studies. How well the dose selection is made can be evaluated only after the
completion of the bioassay. A scientific rationale for dose selection should be clearly articulated
(ILSI, 1997).
In order to obtain the most relevant information from a long-term carcinogenicity study, it
is important to maximize exposure conditions to the test material. At the same time, there is a
need for caution in using excessive high-dose levels that would confound the interpretation of
study results to humans. The middle and lowest doses should be selected to characterize the shape
of the dose-response curve as much as possible. It is important that the doses are adequately
spaced so that the study would provide relevant dose-response data for assessing human hazard
and risk. If the testing of potential carcinogenicity is being combined with an evaluation of
noncancer chronic toxicity, the study should be designed to include one dose that does not elicit
adverse effects.
With regard to the appropriateness of the high dose, an adequate high dose would be one
that produces some toxic effects without either unduly affecting mortality from effects other than
cancer or producing significant adverse effects on the nutrition and health of the test animals
(OECD, 1981; NRC, 1993b). If the test agent does not appear to cause any specific target organ
toxicity or perturbation of physiological function, an adequate high dose would be one that causes
no more than 5%-10% reduction of body weight gain over the lifespan of the animals. The high
dose would be considered inadequate if no toxicity is observed. On the other hand, significant
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increases in mortality from effects other than cancer generally indicate that an adequate high dose
has been exceeded. Other signs of treatment-related toxicity associated with an excessive high
dose may include the following: (a) reduction of body weight gain greater than 10%, (b)
significant increases in abnormal behavioral and clinical signs, (c) significant changes in
hematology or clinical chemistry, (d) saturation of absorption and detoxification mechanisms, or
(e) marked changes in organ weight, morphology, and histopathology. It should be noted that
practical upper limits have been established to avoid the use of excessively high doses in long-
term carcinogenicity studies of environmental chemicals (e.g., 5% of the test substance in the feed
for dietary studies or 1 g/kg of body weight for oral gavage studies [OECD, 1981]).
For dietary studies, weight gain reductions should be evaluated as to whether there is a
palatability problem or an issue with food efficiency; certainly, the latter is a toxic manifestation.
In the case of inhalation studies with respirable particles, evidence of impairment of normal
clearance of particles from the lung should be considered along with other signs of toxicity to the
respiratory airways to determine whether the high exposure concentration has been appropriately
selected. For dermal studies, evidence of skin irritation may indicate that an adequate high dose
has been reached (U.S. EPA, 1989d).
Interpretation of carcinogenicity study results is profoundly affected by study exposure
conditions, especially by inappropriate dose selection. This is particularly important in studies
that are nonpositive for carcinogenicity, since failure to reach a sufficient dose reduces the
sensitivity of the studies. A lack of tumorigenic responses at exposure levels that cause significant
impairment of animal survival may also not be acceptable. In addition, overt toxicity or
inappropriate toxicokinetics due to excessively high doses may result in tumor effects that are
secondary to the toxicity rather than directly attributable to the agent.
There are several possible outcomes regarding the study interpretation of the significance
and relevance of tumorigenic effects associated with exposure or dose levels below, at, or above
an adequate high dose. General guidance is given here that should not be taken as prescriptive;
for each case, the information at hand is evaluated and a rationale should be given for the position
taken.
• Adequate high dose: If an adequate high dose has been utilized, tumor effects are
judged positive or negative depending on the presence or absence of significant tumor
incidence increases, respectively.
• Excessive high dose: If toxicity or mortality is excessive at the high dose,
interpretation depends on the finding of tumors or not.
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(a) Studies that show tumor effects only at excessive doses may be compromised
and may or may not carry weight, depending on the interpretation in the context
of other study results and other lines of evidence. Results of such studies,
however, are generally not considered suitable for dose-response extrapolation if
it is determined that the mode(s) of action underlying the tumorigenic responses
at high doses are not operative at lower doses.
(b) Studies that show tumors at lower doses, even though the high dose is excessive
and may be discounted, should be evaluated on their own merits.
(c) If a study does not show an increase in tumor incidence at a toxic high dose and
appropriately spaced lower doses are used without such toxicity or tumors, the
study is generally judged as negative for carcinogenicity.
• Inadequate high dose: Studies of inadequate sensitivity where an adequate high dose
has not been reached may be used to bound the dose range where carcinogenic effects
might be expected.
Statistical Considerations
The main aim of statistical evaluation is to determine whether exposure to the test agent is
associated with an increase of tumor development. Statistical analysis of a long-term study should
be performed for each tumor type separately. The incidence of benign and malignant lesions of
the same cell type, usually within a single tissue or organ, are considered separately and are
combined when scientifically defensible (McConnell et al., 1986).
Trend tests and pairwise comparison tests are the recommended tests for determining
whether chance, rather than a treatment-related effect, is a plausible explanation for an apparent
increase in tumor incidence. A trend test such as the Cochran-Armitage test (Snedecor and
Cochran, 1967) asks whether the results in all dose groups together increase as dose increases. A
pairwise comparison test such as the Fisher exact test (Fisher, 1950) asks whether an incidence in
one dose group is increased over the control group. By convention, for both tests a statistically
significant comparison is one for whichp <0.05 that the increased incidence is due to chance.
Significance in either kind of test is sufficient to reject the hypothesis that chance accounts for the
result. A statistically significant response may or may not be biologically significant and vice
versa. The selection of a significance level is a policy choice based on a trade-off between the
risks of false positives and false negatives. A significance level of greater or less than 5% is
examined to see if it confirms other scientific information. When the assessment departs from a
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simple 5% level, this should be highlighted in the risk characterization. A two-tailed test or a one-
tailed test can be used. In either case a rationale is provided.
Considerations of multiple comparisons should also be taken into account. Haseman
(1983) analyzes typical animal bioassays testing both sexes of two species and concludes that,
because of multiple comparisons, a single tumor increase for a species-sex-site combination that is
statistically significant at the 1% level for common tumors or 5% for rare tumors corresponds to a
7%-8% significance level for the study as a whole. Therefore, animal bioassays presenting only
one significant result that falls short of the 1% level for a common tumor must be treated with
caution.
Concurrent and Historical Controls
The standard for determining statistical significance of tumor incidence comes from a
comparison of tumors in dosed animals as compared with concurrent control animals. Additional
insights about both statistical and biological significance can come from an examination of
historical control data (Tarone, 1982; Haseman, 1995). Historical control data can add to the
analysis, particularly by enabling identification of uncommon tumor types or high spontaneous
incidence of a tumor in a given animal strain. Identification of common or uncommon situations
prompts further thought about the meaning of the response in the current study in context with
other observations in animal studies and with other evidence about the carcinogenic potential of
the agent. These other sources of information may reinforce or weaken the significance given to
the response in the hazard assessment. Caution should be exercised in simply looking at the
ranges of historical responses because the range ignores differences in survival of animals among
studies and is related to the number of studies in the database.
In analyzing results for uncommon tumors in a treated group that are not statistically
significant in comparison to concurrent controls, the analyst can use the experience of historical
controls to conclude that the result is in fact unlikely to be due to chance. In analyzing results for
common tumors, a different set of considerations comes into play. Generally speaking,
statistically significant increases in tumors should not be discounted simply because incidence
rates in the treated groups are within the range of historical controls or because incidence rates in
the concurrent controls are somewhat lower than average. Random assignment of animals to
groups and proper statistical procedures provide assurance that statistically significant results are
unlikely to be due to chance alone. However, caution should be used in interpreting results that
are barely statistically significant or in which incidence rates in concurrent controls are unusually
low in comparison with historical controls.
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In cases where there may be reason to discount the biological relevance to humans of
increases in common animal tumors, such considerations should be weighed on their own merits
and clearly distinguished from statistical concerns.
When historical control data are used, the discussion needs to address several issues that
affect comparability of historical and concurrent control data. Among these issues are the
following: genetic drift in the laboratory strains, differences in pathology examination at different
times and in different laboratories (e.g., in criteria for evaluating lesions; variations in the
techniques for preparation or reading of tissue samples among laboratories), and comparability of
animals from different suppliers. The most relevant historical data come from the same laboratory
and same supplier, gathered within 2 or 3 years one way or the other of the study under review;
other data should be used only with extreme caution.
Assessment of Evidence of Carcinogenicity from Long-Term Animal Studies
In general, observation of tumor effects under different circumstances lends support to the
significance of the findings for animal carcinogenicity. Significance is a function of the number of
factors present and, for a factor such as malignancy, the severity of the observed pathology. The
following observations add significance to the tumor findings:
• uncommon tumor types;
• tumors at multiple sites;
• tumors by more than one route of administration;
• tumors in multiple species, strains, or both sexes;
• progression of lesions from preneoplastic to benign to malignant;
• reduced latency of neoplastic lesions;
• metastases;
• unusual magnitude of tumor response;
• proportion of malignant tumors; and
• dose-related increases.
These guidelines adopt the science policy position that tumor findings in animals indicate
that an agent may produce such effects in humans. Moreover, the absence of tumor findings in
well-conducted, long-term animal studies in at least two species provides reasonable assurance
that an agent may not be a carcinogenic concern for humans. Each of these is a default
assumption that may be adopted, when appropriate, after evaluation of tumor data and other key
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evidence.
Site Concordance
Site concordance of tumor effects between animals and humans is an issue to be
considered in each case. Thus far, there is evidence that growth control mechanisms at the level
of the cell are homologous among mammals, but there is no evidence that these mechanisms are
site concordant. Moreover, agents observed to produce tumors in both humans and animals have
produced tumors either at the same (e.g., vinyl chloride) or different sites (e.g., benzene) (NRC,
1994). Hence, site concordance is not assumed a priori. On the other hand, certain processes
with consequences for particular tissue sites (e.g., disruption of thyroid function) may lead to an
anticipation of site concordance.
2.2.2.2. Perinatal Carcinogenicity Studies
The objective of perinatal carcinogenesis studies is to determine the carcinogenic potential
and dose-response relationships of the test agent in the developing organism. Some investigators
have postulated that the age of initial exposure to a chemical carcinogen may influence the
carcinogenic response (Vesselinovitch et al., 1979; Rice, 1979; McConnell, 1992). Current
standardized long-term carcinogenesis bioassays generally begin dosing animals at 6-8 weeks of
age and continue dosing for the lifespan of the animal (18-24 months). This protocol has been
modified in some cases to investigate the potential of the test agent to induce transplacental
carcinogenesis or to investigate the potential differences following perinatal and adult exposures;
but currently there is not a standardized protocol for testing agents for carcinogenic effects
following prenatal or early postnatal exposure.
Several cancer bioassay studies have compared adult and perinatal exposures (see
McConnell, 1992; U.S. EPA, 1996a). A review of these reveals that perinatal exposure rarely
identifies carcinogens that are not found in standard animal bioassays. Exposure that is perinatal
sometimes slightly increases the incidence of a given type of tumor. The increase may reflect an
increased length of exposure and a higher dose for the developing organism relative to the adult,
or an increase in sensitivity in some cases. Additionally, exposure that is perinatal through
adulthood sometimes reduces the latency period for tumors to develop in the growing organism
(U.S. EPA, 1996a).
Because the perinatal exposure studies done to date provide only marginal additions to
knowledge as compared with standard bioassay protocols, EPA evaluates the need for such a
study agent-by-agent (U.S. EPA, 1997a,b). Perinatal study data analysis follows the principles
discussed above for evaluating other long-term carcinogenicity studies. When differences in
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responses in perinatal animals compared to adult animals suggest an increased susceptibility or
sensitivity of perinatal or postnatal animals, such as the ones below, a separate evaluation of the
response is prepared:
• a difference in dose-response relationship
• presence of different tumor types
• an earlier onset of tumors
• an increase in the incidence of tumors
An illustrative case study appears in Appendix E.
2.2.2.3. Other Studies
Various intermediate-term studies often use protocols that screen for carcinogenic or
preneoplastic effects, sometimes in a single tissue. Some involve the development of various
proliferative lesions, like foci of alteration in the liver (Goldsworthy et al., 1986). Others use
tumor endpoints, like the induction of lung adenomas in the sensitive strain A mouse (Maronpot
et al., 1986) or tumor induction in initiation-promotion studies using various organs such as the
bladder, intestine, liver, lung, mammary gland, and thyroid (Ito et al., 1992). In these tests, the
selected tissue is, in a sense, the test system rather than the whole animal. Important information
concerning the steps in the carcinogenic process and mode of action can be obtained from
"start/stop" experiments. In these protocols, an agent is given for a period of time to induce
particular lesions or effects, then stopped to evaluate the progression or reversibility of processes
(Todd, 1986; Marsman and Popp, 1994).
Assays in genetically engineered rodents may provide insight into the chemical and gene
interactions involved in carcinogenesis (Tennant et al., 1995). These mechanistically based
approaches involve activated oncogenes that are introduced (transgenic) or tumor suppressor
genes that are deleted (knocked out). If appropriate genes are selected, not only may these
systems provide information on mechanisms, but the rodents typically show tumor development
earlier than the standard bioassay. Transgenic mutagenesis assays also represent a mechanistic
approach for assessing the mutagenic properties of agents as well as developing quantitative
linkages between exposure, internal dose, and mutation related to tumor induction (Morrison and
Ashby, 1994; Sisk et al., 1994; Hayward et al., 1995). These systems use a stable genomic
integration of a lambda shuttle vector that carries a lacl target gene and a lacZ reporter gene.
The support that these studies give to a determination of carcinogenicity rests on their
contribution to the consistency of other evidence about an agent. For instance, benzoyl peroxide
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has promoter activity on the skin, but the overall evidence may be less supportive (Kraus et al.,
1995). These studies also may contribute information about mode of action. One needs to
recognize the limitations of these experimental protocols such as short duration, limited histology,
lack of complete development of tumors, or experimental manipulation of the carcinogenic
process that may limit their contribution to the overall assessment. Generally, their results are
appropriate as aids in the assessment for interpreting other toxicological evidence (e.g., rodent
chronic bioassays), especially regarding potential modes of action. With sufficient validation,
these studies may partially or wholly replace chronic bioassays in the future (Tennant et al., 1995).
2.2.3. Structural Analogue Data
For some chemical classes, there is significant information available on the carcinogenicity
of analogues, largely in rodent bioassays. Analogue effects are instructive in investigating
carcinogenic potential of an agent as well as identifying potential target organs, exposures
associated with effects, and potential functional class effects or modes of action. All appropriate
studies are included and analyzed, whether indicative of a positive effect or not. Evaluation
includes tests in various animal species, strains, and sexes; with different routes of administration;
and at various doses, as data are available. Confidence in conclusions is a function of how similar
the analogues are to the agent under review in structure, metabolism, and biological activity. This
confidence needs to be considered to ensure a balanced position.
2.3. ANALYSIS OF OTHER KEY DATA
The physical, chemical, and structural properties of an agent, as well as data on endpoints
that are thought to be critical elements of the carcinogenic process, provide valuable insights into
the likelihood of human cancer risk. The following sections provide guidance for analyses of
these data.
2.3.1. Physicochemical Properties
Physicochemical properties affect an agent's absorption, tissue distribution
(bioavailability), biotransformation, and degradation in the body and are important determinants
of hazard potential (and dose-response analysis). Properties to analyze include, but are not
limited to, the following: molecular weight, size, and shape; valence state; physical state (gas,
liquid, solid); water or lipid solubility, which can influence retention and tissue distribution; and
potential for chemical degradation or stabilization in the body.
An agent's potential for chemical reaction with cellular components, particularly with
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DNA and proteins, is also important. The agent's molecular size and shape, electrophilicity, and
charge distribution are considered in order to decide whether they would facilitate such reactions.
2.3.2. Structure-Activity Relationships
Structure-activity relationship (SAR) analyses and models can be used to predict
molecular properties, surrogate biological endpoints, and carcinogenicity. Overall, these analyses
provide valuable initial information on agents, may strengthen or weaken concern, and are part of
the weight of evidence.
Currently, SAR analysis is most useful for chemicals and metabolites that are believed to
initiate carcinogenesis through covalent interaction with DNA (i.e., DNA-reactive, mutagenic,
electrophilic, or proelectrophilic chemicals) (Ashby and Tennant, 1991). For organic chemicals,
the predictive capability of SAR analysis combined with other toxicity information has been
demonstrated (Ashby and Tennant, 1994). The following parameters are useful in comparing an
agent to its structural analogues and congeners that produce tumors and affect related biological
processes such as receptor binding and activation, mutagenicity, and general toxicity (Woo and
Arcos, 1989):
• nature and reactivity of the electrophilic moiety or moieties present;
• potential to form electrophilic reactive intermediate(s) through chemical,
photochemical, or metabolic activation;
• contribution of the carrier molecule to which the electrophilic moiety(ies) is attached;
• physicochemical properties (e.g., physical state, solubility, octanol-water partition
coefficient, half-life in aqueous solution);
• structural and substructural features (e.g., electronic, stearic, molecular geometric);
• metabolic pattern (e.g., metabolic pathways and activation and detoxification ratio);
and
• possible exposure route(s) of the agent.
Suitable SAR analysis of non-DNA-reactive chemicals and of DNA-reactive chemicals
that do not appear to bind covalently to DNA requires knowledge or postulation of the probable
mode(s) of action of closely related carcinogenic structural analogues (e.g., receptor-mediated,
cytotoxicity-related). Examination of the physicochemical and biochemical properties of the
agent may then provide the rest of the information needed in order to make an assessment of the
likelihood of the agent's activity by that mode of action.
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2.3.3. Comparative Metabolism and Toxicokinetics
Studies of the absorption, distribution, biotransformation, and excretion of agents permit
comparisons among species to assist in determining the implications of animal responses for
human hazard assessment, supporting identification of active metabolites, identifying changes in
distribution and metabolic pathway or pathways over a dose range, and making comparisons
among different routes of exposure.
If extensive data are available (e.g., blood/tissue partition coefficients and pertinent
physiological parameters of the species of interest), physiologically based pharmacokinetic models
can be constructed to assist in a determination of tissue dosimetry, species-to-species
extrapolation of dose, and route-to-route extrapolation (Connolly and Andersen, 1991; see
Section 3.2.2). If it is not contrary to available data, it is assumed as a default that toxicokinetic
and metabolic processes are qualitatively comparable between species. Discussion of the defaults
regarding quantitative comparison and their modifications appears in Chapter 3.
The qualitative question of whether an agent is absorbed by a particular route of exposure
is important for weight-of-evidence classification, discussed in Section 2.7.1. Decisions whether
route of exposure is a limiting factor on expression of any hazard, in that absorption does not
occur by a route, are based on studies in which effects of the agent, or its structural analogues,
have been observed by different routes, on physical-chemical properties, or on toxicokinetics
studies.
Adequate metabolism and pharmacokinetic data can be applied toward the following as
data permit. Confidence in conclusions is enhanced when in vivo data are available.
• Identifying metabolites and reactive intermediates of metabolism and determining
whether one or more of these intermediates are likely to be responsible for the
observed effects. This information on the reactive intermediates will appropriately
focus SAR analysis, analysis of potential modes of action, and estimation of internal
dose in dose-response assessment (D'Souza et al., 1987; Krewski et al., 1987).
• Identifying and comparing the relative activities of metabolic pathways in animals with
those in humans as well as different ages. This analysis can provide insights for
extrapolating results of animal studies to humans.
• Describing anticipated distribution within the body and possibly identifying target
organs. Use of water solubility, molecular weight, and structure analysis can support
qualitative inferences about anticipated distribution and excretion. In addition,
describing whether the agent or metabolite of concern will be excreted rapidly or
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slowly or will be stored in a particular tissue or tissues to be mobilized later can
identify issues in comparing species and formulating dose-response assessment
approaches.
• Identifying changes in toxicokinetics and metabolic pathways with increases in dose.
These changes may result in important differences in disposition of the agent or its
generation of active forms of the agent between high and low dose levels. These
studies play an important role in providing a rationale for dose selection in
carcinogenicity studies.
• Identifying and comparing metabolic process differences by age, sex, or other
characteristic so that sensitive subpopulations can be recognized. For example,
metabolic capacity with respect to P450 enzymes in newborn children is extremely
limited compared to adults, so that a requirement for metabolic activation of a
carcinogen will limit its effect in young, whereas a requirement for metabolic
deactivation will result in increased sensitivity of this subpopulation (Cresteil, 1998).
A variety of changes in toxicokinetics and physiology occur from fetal to post-
weaning, to young child. Any of these may make a difference to risk (Renwick, 1998)
• Determining bioavailability via different routes of exposure by analyzing uptake
processes under various exposure conditions. This analysis supports identification of
hazards for untested routes. In addition, use of physicochemical data (e.g., octanol-
water partition coefficient information) can support an inference about the likelihood
of dermal absorption (Flynn, 1990).
In all of these areas, attempts are made to clarify and describe as much as possible the
variability to be expected because of differences in species, sex, age, and route of exposure. The
analysis takes into account the presence of subpopulations of individuals who are particularly
vulnerable to the effects of an agent because of toxicokinetic or metabolic differences (genetically
or environmentally determined) (Bois et al., 1995), and is a special emphasis for assessment of
risks to children.
2.3.4. Toxicological and Clinical Findings
Toxicological findings in experimental animals and clinical observations in humans are an
important resource to the cancer hazard assessment. Such findings provide information on
physiological effects and effects on enzymes, hormones, and other important macromolecules, as
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well as on target organs for toxicity. Given that the cancer process represents defects in terminal
differentiation, growth control, and cell death, developmental studies of agents may provide an
understanding of the activity of an agent that carries over to cancer assessment. Toxicity studies
in animals by different routes of administration support comparison of absorption and metabolism
by those routes. Data on human variability in standard clinical tests may provide insight into the
range of human sensitivity and common mechanisms to agents that affect the tested parameters.
2.3.5. Events Relevant to Mode of Carcinogenic Action
Information on the biochemical and biological changes that precede tumor development
(which includes but is not limited to mutagenesis, increased cell proliferation, inhibition of
programmed cell death, and receptor activation) may provide important information in
determining whether a cancer hazard exists and may help inform the dose-response relationship
below the range of observable tumor response. Because cancer is the result of a series of genetic
defects in genes controlling cell growth, division, and differentiation (Vogelstein et al., 1988), the
ability of an agent to affect genes or gene expression is of obvious importance in evaluating its
influence on the carcinogenic process. Initial and key questions to examine are: Does the agent (or
its metabolite) interact directly with and mutate DNA to bring about changes in gene expression?
Does the agent bring about effects on gene expression via other processes? Furthermore,
carcinogenesis involves a complex series and interplay of events that alter the signals a cell
receives from its extracellular environment to promote growth. Many, but not all, mutagens are
carcinogens, and some, but not all, agents that induce cell proliferation lead to tumor
development. Thus, understanding the range of key influences that the chemical may have on the
carcinogenic process is essential for evaluating mode of action. Endpoints that provide insight
into an agent's ability to alter genes and gene expression and other features of an agent's potential
mode of carcinogenic action are discussed below.
2.3.5.1. Direct DNA Reactive Effects
It is well known that many carcinogens are electrophiles that interact with DNA, resulting
in DNA adducts and breakage (referred to in these guidelines as direct DNA effects). Following
DNA replication, these DNA lesions can be converted into mutations and stable cytogenetic
alterations, which then may initiate and contribute to the carcinogenic process (Shelby and Zeiger,
1990; Tinwell and Ashby, 1991). Thus, studies of mutations and other genetic lesions continue to
be important in the assessment of potential human cancer hazard and in the understanding of an
agent's mode of carcinogenic action. EPA has published testing guidelines for detecting the
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ability of an agent to damage DNA and produce mutations and chromosomal aberrations. Briefly,
standard tests for gene mutations in bacteria and mammalian cells in vitro and in vivo, and for
structural chromosomal aberrations in vitro and in vivo are important examples of relevant
methods. New molecular approaches such as mouse mutations and cancer transgenic models are
providing a means to examine mutation at tissue sites where the tumor response is observed
(Heddle and Swiger, 1996). Additionally, continued improvements in fluorescent-based
chromosome staining methods (FISH, fluorescent in situ hybridization) will allow the detection of
specific chromosomal abnormalities in relevant target tissues (Tucker and Preston, 1998).
Endpoints indicative of DNA damage but not measures of mutation per se, such as DNA
adducts or strand breakage, can be detected in relevant target tissues and thus contribute to
evaluating an agent's mutagenic potential. Evidence of chemical-specific DNA adducts (e.g.,
reactions at oxygen sites in DNA bases or with ring nitrogens of guanine and adenine) provides
information on a mutagen's ability to directly interact with DNA (La and Swenberg, 1996). It
should be noted that an increase in DNA binding shown with a radioactive label incorporated in
the chemical (e.g., C14) may reflect a direct DNA reactive mechanism, but needs to be examined
because the label may reflect reuse of C14 in the synthesis of DNA rather than binding. Some
planar molecules (e.g., 9-aminoacridine) intercalate between base pairs of DNA, which results in a
physical distortion in DNA that may lead to mutations when DNA replicates. As discussed
below, some carcinogens do not interact directly with DNA, but can produce increases in
endogenous levels of DNA adducts (e.g., 8-hydroxyguanine) by indirect mechanisms.
2.3.5.2. Indirect DNA Effects or Other Effects on Genes/Gene Expression
Although some carcinogens may result in an elevation of mutations or cytogenetic
anomalies as detected in standard assays, they may do so by indirect mechanisms. These effects
may be brought about by chemical-cell interactions rather than the chemical (or its metabolite)
directly interacting with DNA. An increase in mutations might be due to cytotoxic exposures
causing regenerative proliferation or to mitogenic influences (Cohen and Ellwein, 1990).
Increased cell division may elevate mutation by clonal expansion of initiated cells or by increasing
the number of genetic errors by rapid cell division and reduced time for DNA repair. Some agents
might result in an elevation of mutations by interfering with the enzymes involved in DNA repair
and recombination (Barrett and Lee, 1992). Damage to certain critical DNA repair genes or other
genes (e.g., the p53 gene) may result in genomic instability, which predisposes cells to further
genetic alterations and increases the probability of neoplastic progression (Harris and Hollstein,
1993; Levine, 1994). Likewise, DNA repair processes may be saturated at certain doses of a
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chemical, and thus result in an elevation of genetic alterations. Programmed cell death (apoptosis)
can potentially be blocked by an agent, thereby permitting replication of cells carrying genetic
errors. For example, peroxisome proliferators may act by suppressing apoptotic pathways
(Shulte-Hermann et al., 1993; Bayly et al., 1994). At certain doses an agent may also generate
reactive oxygen species that produce oxidative damage to DNA and other important
macromolecules (Kehrer, 1993; Clayson et al., 1994; Chang et al., 1988). The role of these
adducts, attributable to oxidative damage (e.g., 8-hydroxyguanine), in tumorigenesis is currently
unclear.
Several carcinogens have been shown to induce aneuploidy (Gibson et al., 1995; Barrett,
1992). The loss or gain of chromosomes (i.e., aneuploidy) can result in the loss of heterozygosity
or genomic instability (Fearon and Vogelstein, 1990; Cavenee et al., 1986). Agents that cause
aneuploidy typically interfere with the normal process of chromosome segregation by interacting
with non-DNA targets such as the proteins needed for chromosome movement. All tumors
(except leukemias and lymphomas) are aneuploid, but whether this is the cause or the effect of
tumorigenesis is not clear. Thus, it is important to understand whether the agent induces
aneuploidy as a key early event in the carcinogenic process or is necessary for tumor progression.
It is possible for an agent to alter gene expression by transcriptional, translational, or post-
translational modifications (Barrett, 1995). For example, perturbation of DNA methylation
patterns may cause effects that contribute to carcinogenesis (Jones, 1986; Goodman and Counts,
1993; Holliday, 1987; Chuang et al., 1996). Overexpression of genes by DNA amplification has
been observed in certain tumors (Vainio et al., 1992). Gene amplification may result from
disproportionate DNA replication. Other mechanisms of altering gene expression may involve
cellular reprogramming through hormonal or receptor-mediated mechanisms (Ashby et al., 1994;
Barrett, 1992).
Both cell proliferation and programmed cell death are mandatory for the maintenance of
homeostasis in normal tissue, and when altered become important elements of the carcinogenic
process. The balance between the two directly affects the survival and growth of initiated cells, as
well as preneoplastic and tumor cell populations (i.e., increase in cell proliferation or decrease in
cell death) (Bellamy et al., 1995; Cohen and Ellwein, 1990, 1991; Cohen et al., 1991). Thus,
measures of these events contribute to the weight of the evidence for cancer hazard and to mode-
of-action understanding. In studies of proliferative effects, distinctions should be made between
mitogenesis and regenerative proliferation (Cohen and Ellwein, 1990, 1991; Cohen et al., 1991).
In applying information from studies on cell proliferation and apoptosis to risk assessment, it is
important to identify the tissues and target cells involved, to measure effects in both normal and
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neoplastic tissue, to distinguish between apoptosis and necrosis, and to determine the dose that
affects these processes. Gap-junctional intercellular communication is believed to play a role in
tissue and organ development and in the maintenance of a normal cellular phenotype within
tissues. A growing body of evidence suggests that chemical interference with gap-junctional
intercellular communication is a contributing factor in tumor development (Swierenga and
Yamasaki, 1992; Yamasaki, 1995).
2.3.5.3. Experimental Considerations in Evaluating Data on Precursor Events
Most testing schemes for mutagenicity and other short-term assays were designed for
hazard identification purposes; thus, these assays are generally conducted using acute exposures.
For data on "precursor steps" to be useful in informing the dose-response curve for tumor
induction below the level of observation, it is important that data come from in vivo studies where
exposure is repeated or given over an extended period of time. Although consistency of results
across different assays and animal models provides a stronger basis for drawing conclusions, it is
desirable to have data on the precursor event in the same target organ, sex, animal strain, and
species as the tumor data. In evaluating an agent's mode of action, it is usually not sufficient to
determine that some event commences upon dosing. It is important to understand whether it is a
causal event that plays a key role in the process that leads to tumor development, versus an effect
of the cancer process itself or simply an associated event.
2.3.5.4. Judging Data
Criteria that are applicable for judging the adequacy of mechanistically based data include
the following:
• mechanistic relevance of the data to carcinogenicity,
• number of studies of each endpoint,
• consistency of results in different test systems and different species,
• similar dose-response relationships for tumor and mode of action-related effects,
• tests conducted in accordance with generally accepted protocols, and
• degree of consensus and general acceptance among scientists regarding interpretation
of the significance and specificity of the tests.
Although important information can be gained from in vitro test systems, a higher level of
confidence is generally given to data that are derived from in vivo systems, particularly those
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results that show a site concordance with the tumor data.
2.4. BIOMARKER INFORMATION
Various endpoints can serve as biological markers of events in biological systems or
samples. In some cases, these molecular or cellular effects (e.g., DNA or protein adducts,
mutation, chromosomal aberrations, levels of thyroid stimulating hormone) can be measured in
blood, body fluids, cells, and tissues to serve as biomarkers of exposure in both animals and
humans (Callemen et al., 1978; Birner et al., 1990). As such, they can do the following:
• act as an internal surrogate measure of chemical dose, representing as appropriate,
either recent (e.g., serum concentration) or accumulated (e.g., hemoglobin adducts)
exposure;
• help identify doses at which elements of the carcinogenic process are operating;
• aid in interspecies extrapolations when data are available from both experimental
animal and human cells; and
• under certain circumstances, provide insights into the possible shape of the dose-
response curve below levels where tumor incidences are observed (e.g., Choy, 1993).
Genetic and other findings (like changes in proto-oncogenes and tumor suppressor genes
in preneoplastic and neoplastic tissue or, possibly, measures of endocrine disruption) can indicate
the potential for disease and as such serve as biomarkers of effect. They, too, can be used in
different ways:
• The spectrum of genetic changes in proliferative lesions and tumors following chemical
administration to experimental animals can be determined and compared with those in
spontaneous tumors in control animals, in animals exposed to other agents of varying
structural and functional activities, and in persons exposed to the agent under study.
• They may provide a linkage to tumor response.
• They may help to identify subpopulations of individuals who may be at an elevated risk
for cancer, e.g., cytochrome P450 2D6/debrisoquine sensitivity for lung cancer
(Caporaso et al., 1989) or inherited colon cancer syndromes (Kinzler et al., 1991;
Peltomaki et al., 1993).
• As with biomarkers of exposure, it may be justified in some cases to use these
endpoints for dose-response assessment or to provide insight into the potential shape
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of the dose-response curve at doses below those at which tumors are induced
experimentally.
In applying biomarker data to cancer assessment (particularly assessments based on
epidemiologic data), one should consider the following:
• routes of exposure,
• exposure to mixtures,
• time after exposure,
• sensitivity and specificity of biomarkers, and
• dose-response relationships.
2.5. MODE OF ACTION-GENERAL CONSIDERATIONS AND FRAMEWORK FOR
ANALYSIS
2.5.1. General Considerations
The interaction of the biology of the organism and the chemical properties of the agent
determine whether there is an adverse effect. Thus, mode-of-action analysis is based on physical,
chemical, and biological information that helps to explain key events5 in an agent's influence on
development of tumors. The entire range of information developed in the assessment is reviewed
to arrive at a reasoned judgment. An agent may work by more than one mode of action both at
different sites and at the same tumor site. It is felt that at least some information bearing on mode
of action (e.g., SAR, screening tests for mutagenicity) is present for most agents undergoing
assessment of carcinogenicity, even though certainty about exact molecular mechanisms may be
rare.
Inputs to mode-of-action analysis include tumor data in humans, animals, and among
structural analogues as well as the other key data. The more complete the data package and
generic knowledge about a given mode of action, the more confidence one has and the more one
can replace or refine default science policy positions with relevant information. Making reasoned
judgments is generally based on a data-rich source of chemical, chemical class, and tumor type-
specific information. Many times there will be conflicting data and gaps in the information base;
one must carefully evaluate these uncertainties before reaching any conclusion.
2A "key event" is an empirically observable, precursor step that is itself a necessary element of the
mode of action, or is a marker for such an element.
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In making decisions about potential modes of action and the relevance of animal tumor
findings to humans (Ashby et al., 1990), very often the results of chronic animal studies may give
important clues. Some of the important factors to review include the following:
• tumor types, e.g., those responsive to endocrine influence or those produced by
reactive carcinogens (Ashby and Tennant, 1991);
• number of tumor sites, sexes, studies, and species affected or unaffected (Tennant,
1993);
• influence of route of exposure, spectrum of tumors, and local or systemic sites;
• target organ or system toxicity, e.g., urinary chemical changes associated with stone
formation, effects on immune surveillance;
• presence of proliferative lesions, e.g., hepatic foci, hyperplasias;
• progression of lesions from preneoplastic to benign to malignant with dose and time;
• ratio of malignant to benign tumors as a function of dose and time;
• time of appearance of tumors after commencing exposure;
• tumors invading locally, metastasizing, producing death;
• tumors at sites in laboratory animals with high or low spontaneous historical incidence;
• biomarkers in tumor cells, both induced and spontaneous, e.g., DNA or protein
adducts, mutation spectra, chromosome changes, oncogene activation; and
• shape of the dose response in the range of tumor observation, e.g., linear vs. profound
change in slope.
Some of the myriad of ways that information from chronic animal studies influences mode-
of-action judgments include the following. Multisite and multispecies tumor effects are often
associated with mutagenic agents. Tumors restricted to one sex/species may suggest an influence
restricted to gender, strain, or species. Late onset of tumors that are primarily benign or are at
sites with a high historical background incidence or show reversal of lesions on cessation of
exposure may point to a growth-promoting mode of action. The possibility that an agent may act
differently in different tissues or have more than one mode of action in a single tissue must also be
kept in mind.
Simple knowledge of sites of tumor increase in rodent studies can give preliminary clues
as to mode of action. Experience at the National Toxicology Program (NTP) indicates that
substances that are DNA reactive and produce gene mutations may be unique in producing
tumors in certain anatomical sites, while tumors at other sites may arise from both mutagenic or
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nonmutagenic influences (Ashby and Tennant, 1991; Huff et al., 1991).
Effects on tumor sites in rodents and other mode-of-action information has been explored
for certain agents (Alison et al., 1994; Clayson, 1989; ECETOC, 1991; MacDonald et al., 1994;
McClain, 1994; Tischler et al., 1991; ILSI, 1995; Cohen and Ellwein, 1991; FASEB, 1994; Havu
et al., 1990; U.S. EPA, 1991c; Li et al., 1987; Grasso and Hinton, 1991; Larson et al., 1994;
IARC, 1990; Jack et al., 1983; Stitzel et al., 1989; Ingram and Grasso, 1991; Bus and Popp,
1987; Prahalada et al., 1994; Yamada et al., 1994; Hill et al., 1989; Burek et al., 1988).
2.5.2. Evaluating a Postulated Mode of Action
Peer Review
This section contains a framework for evaluating a postulated mode of action. In reaching
conclusions, the question of "general acceptance" of a mode of action will be tested as part of the
independent peer review that EPA obtains for its assessment and conclusions. In some cases the
mode of action may have already been established by development of a large body of research
information and characterization of the phenomenon over time. In some cases there will have
been development of an Agency policy, e.g., male rat thyroid disruption, or a series of previous
assessments in which both the mode of action and its applicability to particular cases has been
explored, e.g., urinary bladder stones. If so, the assessment and its peer review can be focused on
the evidence that a particular agent acts in this mode.
In other cases, the mode of action previously may not have been the subject of an Agency
document. If so, the data to support both the mode of action and the activity of the agent with
respect to it will be the subjects of EPA assessment and subsequent peer review.
Use of the Framework
The framework supports a full analysis of mode-of-action information, but can also be
used as a screen to decide whether sufficient information is available to evaluate or the data gaps
are too substantial to justify further analysis. Mode-of-action conclusions are used to address the
question of human relevance of animal tumor responses, to address differences in anticipated
response among humans such as between children and adults or men and women, and as the basis
of decisions about the anticipated shape of the dose-response relationship. Guidance on the latter
appears in Section 3.
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2.5.3. Framework for Evaluating a Postulated Carcinogenic Mode(s) of Action
This framework is intended to be an analytic tool for judging whether available data
support a mode of carcinogenic action postulated for an agent. It is based upon considerations
for causality in epidemiologic investigations originally articulated by Hill, but later modified by
others and extended to experimental studies. The original Hill criteria were applied to
epidemiologic data, while this framework is applied to a much wider assortment of experimental
data, so it retains the basic principles of Hill but is much modified in content.
A mode of action is composed of key events and processes starting with the interaction of
an agent with a cell, through operational and anatomical changes, resulting in cancer formation.
"Mode" of action is contrasted with "mechanism" of action, which implies a more detailed,
molecular description of events than is meant by mode of action. There are many examples of
possible modes of carcinogenic action, such as mutagenicity, mitogenesis, inhibition of cell death,
cytotoxicity with reparative cell proliferation, and immune suppression. All pertinent studies are
reviewed in analyzing a mode of action, and an overall weighing of evidence is performed, laying
out the strengths, weaknesses, and uncertainties of the case as well as potential alternative
positions and rationales. Identifying data gaps and research needs is also part of the assessment.
To show that a postulated mode of action is operative, it is generally necessary to outline
the sequence of events leading to cancer, to identify key events that can be measured, and to
weigh information to determine whether there is a causal relationship between events and cancer
formation. In no case will it be expected that the complete sequence is known at the molecular
level. Instead, empirical observations made at different levels of biological organization are
analyzed: biochemical, cellular, physiological, tissue, organ, and system levels.
Several important points should be kept in mind when working with the framework:
• The topics listed for analysis should not be regarded as a checklist of necessary
"proofs." The judgment whether a postulated mode of action is supported by available
data takes account of the analysis as a whole.
• The framework provides a structure for organizing the facts upon which conclusions
as to mode of action rest. The purpose of using the framework is to make analysis
transparent and allow the reader to understand the facts and reasoning behind a
conclusion.
• The framework does not dictate an answer. The weight of evidence that is sufficient to
support a decision about a mode of action may be less or more depending on the
purpose of the analysis, e.g., screening, research needs identification, or full risk
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assessment. To make the reasoning transparent, the purpose of the analysis ought to
be made apparent to the reader.
• Toxicokinetic studies may contribute to mode-of-action analysis by identifying the
active form of an agent that is central to the mode of action. Apart from contributing
in this way, toxicokinetics studies may reveal effects of saturation of metabolic
processes. These are not considered key events in a mode of action, but are given
separate consideration in assessing dose metrics and potential nonlinearity of the dose-
response relationship.
• Generally, "sufficient" support is a matter of scientific judgment in the context of the
requirements of the decision maker or in the context of science policy guidance
regarding a certain mode of action.
• While a postulated mode of action may be supported for a described response in a
specific tissue, it may not explain other tumor responses observed. The latter will need
separate consideration in hazard and dose-response assessment.
It is anticipated that in a risk assessment document, the analysis of a postulated mode of
action will be presented before or with the characterization of an agent's potential hazard to
humans.
2.5.3.1. Content of the Framework
The framework analysis begins with a summary description of the postulated mode of
action for a tumor type. (Each postulated mode of action requires separate analysis.) This is
followed by topics for analysis and presentation in a convenient order. For illustration, the
explanation of each topic includes typical questions to be addressed to the available empirical data
and experimental observations anticipated to be pertinent. The latter will vary from case to case.
For a particular mode of action, certain observations may be established as essential in practice or
policy, e.g., measures of thyroid hormone levels in supporting thyroid hormone elevation as a key
event in carcinogenesis. A conclusion and an analysis of human relevance including
subpopulations are the the final parts of the analysis.
1. Summary Description of Postulated Mode of Action
This description briefly explains the sequence of events and processes that are considered
to lead to cancer formation. For example, for thyroid disruption and thyroid follicular cell tumors:
Thyroid hormone production is regulated by actions of the hypothalamus,
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pituitary, and thyroid gland. Homeostasis of thyroid hormone is maintained by
a feedback loop between the hypothalamus and pituitary and the thyroid gland.
The hypothalamus produces thyrotrophin reducing hormone (TRH), which
stimulates the pituitary to produce thyroid stimulating hormone (TSH) which,
in turn, stimulates the thyroid to produce thyroid hormone. The hypothalamus
and pituitary respond to high levels of circulating thyroid hormone by
suppressing TRH and TSH production, and to a low level by increasing them.
The mode of action considered is continuous elevation of TSH levels that
stimulates the thyroid gland to deplete its stores of thyroid hormone and
continues to push production resulting in hypertrophy of the production cells
(follicular cells) leading to hyperplasia, nodular hyperplasia, and, eventually,
tumors of these cells. In rats, the chain of events may be induced by direct
effects on hormone synthesis or by metabolic removal of circulating hormone.
2. "Identification of key events " is a consideration devised for this framework. A "key
event" is an empirically observed precursor step consistent with a mode of action. In order to
judge how well data support involvement of an event in carcinogenic processes, the experimental
definition of the event or events must be clear and repeatable. To support an association,
experiments need to define and measure an event consistently.
• Can a list of events be identified that are key to the carcinogenic process?
• Are the events well defined?
Pertinent observations: e.g., increased cell growth, organ weight, histology, proliferation assays,
hormone or other protein perturbations, receptor-ligand changes, DNA or chromosome effects,
cell cycle effects.
3. "Strength, consistency, specificity of association A statistically significant
association between events and a tumor response observed in well-conducted studies is
supportive of causation. Consistent observations in a number of such studies with differing
experimental designs increases that support, since different designs may reduce unknown biases.
Studies showing "recovery," i.e., absence or reduction of carcinogenicity when the event is
blocked or diminished, are particularly important tests of the association. Specificity of the
association, without evidence of other modes of action, strengthens a causal conclusion.
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• What is the level of statistical and biological significance for each event and for
cancer?
• Do independent studies and different experimental hypothesis-testing approaches
produce the same associations?
• Does the agent produce effects other than postulated?
• Is the key event associated with precursor lesions?
Pertinent observations: e.g., tumor response associated with events (site of action logically relates
to event[s]), precursor lesions associated with events, initiation-promotion studies, stop/recovery
studies.
4. "Dose-response relationship If a key event and tumor endpoints increase with dose,
a causal association can be strengthened. Dose-response associations of the key event with other
precursor events can add further strength. Difficulty arises when an event is not causal, but
accompanies the process generally. Dose-response studies coupled with mechanistic studies can
assist in clarifying these relationships.
• What are the correlations among doses producing events and cancer?
Pertinent observations: e.g., 2-year bioassay observation of lesions correlated with observations of
hormone changes and the same lesions in shorter term studies or in interim sacrifice.
5. "Temporal relationship If an event is a cause of tumorigenesis, it must precede
tumor appearance. An event may also be observed contemporaneously or after tumor
appearance; these observations may add to the strength of association, but not to the temporal
association.
• What is the ordering of events that underlie the carcinogenic process?
• Is this ordering consistent among independent studies?
Pertinent observations: Studies of varying duration observing the temporal sequence of
events and tumorigenicity.
6. "Biologicalplausibility and coherence The postulated mode of action and the
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events that are part of it need to be based on current understanding of the biology of cancer to be
accepted. If the body of information under scrutiny is consistent with other examples (including
structurally related agents) for which the postulated mode of action is accepted, the case is
strengthened. Since some modes of action can be anticipated to evoke effects other than cancer,
the available toxicity database on noncancer effects can contribute to this evaluation, e.g.,
reproductive effects of certain hormonal disturbances.
• Is the mode of action consistent with what is known about carcinogenesis in general
and for the case specifically?
• Are carcinogenic effects and events consistent across structural analogues?
• Is the database on the agent internally consistent in supporting the purported mode of
action, including relevant noncancer toxicities?
Pertinent observations: Scientific basis for considering a postulated mode of action generally,
given current state of knowledge of carcinogenic processes; previous examples of data sets
showing the mode of action; data sets on analogues; coherence of data in this case from cancer
and noncancer toxicity studies.
7. "Other modes of action This discussion covers alternative modes of action for the
tumor response considered and whether they are supported by the data. In addition, it provides a
place to discuss other tumor observations that may be arising from a different mode of action than
postulated.
8. "Conclusion This is a brief conclusion and rationale as to whether the postulated
mode of action is supported, also reflecting the purpose of the evaluation. The conclusion that a
mode of action is supported is stronger as more of the above topic analyses point in the same
direction, and weaker as fewer do so. The testing of the mode of action hypothesis by various
experimental approaches with the same result creates a stronger basis for conclusions.
Characteristics of strength of support include data showing that all key events are in sequence
prior to tumor formation, dose and timing are consistent with the sequence, and reversal or
reduction of key events and effects occurs upon cessation of dosing. The conclusion should
address whether key event or associated metabolic information allows identification of rate-
limiting measures of either the mode of action or of toxicokinetics.
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9. "Human relevance, including subpopulations " : This is an analysis of data on the
question whether a mode of action found to be operative in animals is also operative in humans
and whether any human subpopulation is apt to qualitatively respond to the mode of action
differently than the general population. Relevance to humans of animal responses is the default
assumption since metazoans appear to share the basic modes of carcinogenic action.
When sufficient information is developed in mature animals to show a mode of action for a
specific tumor type, an evaluation will be made of whether the mode of action is qualitatively
applicable to children (including infants and fetuses), i.e., same sequence of key events is
anticipated to be involved. Ideally we would have data pertinent to the question with respect to
the agent under assessment. In the absence of such data, a cogent biological rationale needs to
be developed regarding whether the mode of action is applicable to children. For the latter, the
evaluation would cover the scientific information at large, including such considerations as
age-related similarities and differences in the occurrence of the specific tumor type in the U.S.
population, in occurrence of identified key events of the mode of action, in pertinent biochemical,
physiological and toxicological processes, and in metabolism and excretion of the agent.
Examples are given in case examples for chemicals T and Z in Appendix D. Based on the
similarities of tumors following exposure to radiation, pharmaceuticals and viruses, from a
qualitative standpoint, it may be anticipated that the same kind of tumors may develop following
childhood or adult exposure to environmental chemicals. However, when there are no
agent-specific data or there is not a cogent rationale supporting the comparability between
responses in children and adults, the mode of action will not be considered to be applicable for
children. It should also be noted that from a quantitative perspective, the same key events may
lead to greater or lesser occurrence at different agents due to toxicokinetic and exposure
considerations. These considerations need separate evaluation and may result in separate risk
estimates for the young or for that portion of a lifetime.
2.6. WEIGHT-OF-EVIDENCE EVALUATION FOR POTENTIAL HUMAN
CARCINOGENICITY
A weight-of-evidence evaluation is a collective evaluation of all pertinent information so
that the full impact of biological plausibility and coherence is adequately considered.
Identification and characterization of human carcinogenicity is based on human and experimental
data, the nature, advantages, and limitations of which have been discussed in the preceding
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sections.
The subsequent sections outline: (1) the basics of weighing individual lines of evidence
and combining the entire body of evidence to make an informed judgment, and (2) classification
descriptors of cancer hazard.
2.6.1. Weight-of-Evidence Analysis
Judgment about the weight of evidence involves considerations of the quality and
adequacy of data and consistency of responses induced by the agent in question. The weight-of-
evidence judgment requires combined input of relevant disciplines. Initial views of one kind of
evidence may change significantly when other information is brought to the interpretation. For
example, a positive animal carcinogenicity finding may be diminished by other key data; a weak
association in epidemiologic studies may be bolstered by consideration of other key data and
animal findings. Factors typically considered are illustrated in figures below. Generally, no single
weighing factor on either side determines the overall weight. The factors are not scored
mechanically by adding pluses and minuses; they are judged in combination.
Human Evidence
Analyzing the contribution of evidence from a body of human data requires examining
available studies and weighing them in the context of well-accepted criteria for causation (see
Section 2.2.1). A judgment is made about how closely the studies satisfy these criteria,
individually and jointly, and how far they deviate from them. Existence of temporal relationships,
consistent results in independent studies, strong association, reliable exposure data, presence of
dose-related responses, freedom from biases and confounding factors, and high level of statistical
significance are among the factors leading to increased confidence in a conclusion of causality.
Generally, the weight of human evidence increases with the number of adequate studies
that show comparable results on populations exposed to the same agent under different
conditions. The analysis takes into account all studies of high quality, whether showing positive
associations or null results, or even protective effects. In weighing positive studies against null
studies, possible reasons for inconsistent results should be sought, and results of studies that are
judged to be of high quality are given more weight than those from studies judged to be
methodologically less sound. See Figure 2-1.
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Increase weight
Number of independent studies with
consistent results
Most causal criteria satisfied:
Temporal relationship
Strong association
Reliable exposure data
Dose-response relationship
Freedom from bias and confounding
Biological plausibility
High statistical significance
Decrease weight
Few studies
Equally well-designed and conducted studies
with null results
Few causal criteria satisfied
Figure 2-1. Factors for weighing human evidence.
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Generally, no single factor is determinative. For example, strength of association is one of
the causal criteria. A strong association (i.e., a relatively large risk) is more likely to indicate
causality than a weak association. However, finding of a large excess risk in a single study must
be balanced against the lack of consistency as reflected by null results from other equally well-
designed and well-conducted studies. In this situation, the positive association of a single study
may either suggest the presence of chance, bias, or confounding, or reflect different exposure
conditions. On the other hand, evidence of weak but consistent associations across several
studies suggests either causality or that the same confounder may be operating in all of these
studies.
Animal Evidence
Evidence from long-term or other carcinogenicity studies in laboratory animals constitutes
the second major class of information bearing on carcinogenicity. See Figure 2-2. As discussed
in Section 2.2.2, each relevant study must be reviewed and evaluated as to its adequacy of design
and conduct as well as the statistical significance and biological relevance of its findings. Factors
that usually increase confidence in the predictivity of animal findings are those of (1) multiplicity
of observations in independent studies; (2) severity of lesions, latency, and lesion progression; and
(3) consistency in observations.
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Increase weight Decrease weight
Number of independent studies with
Single study
consistent results
Inconsistent results
Same site across species, structural
analogues
Multiple observations
Single site/species/sex
Species
Sites
Sexes
Severity and progression of lesions
Benign tumors only
Early-in-life tumors/malignancy
Dose-response relationships
High background of incidence tumors
Lesion progression
Uncommon tumor
Route of administration like human
Route of administration unlike human
exposure
exposure
A
Figure 2-2. Factors for weighing animal evidence.
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1 Other Key Data
2 Additional information bearing on the qualitative assessment of carcinogenic potential may
3 be gained from comparative pharmacokinetic and metabolism studies, genetic toxicity studies,
4 SAR analysis, and other studies of an agent's properties. See Figure 2-3. Information from these
5 studies helps to elucidate potential modes of action and biological fate and disposition. The
6 knowledge gained supports interpretation of cancer studies in humans and animals and provides a
7 separate source of information about carcinogenic potential.
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Increase weight Decrease weight
A rich set of other key data are available
Physicochemical data
Data indicate reactivity with macromolecules
Structure-activity relationships support
hazard potential
Comparable metabolism and toxicokinetics
between species
Toxicological and human clinical data support
tumor findings
Biomarker data support attribution of
effects to agent
Mode-of-action data support causal
interpretation of human evidence or
relevance of animal evidence
Few or poor data
or
Inadequate data necessitate use of default
assumptions
or
Data show that animal findings are not
relevant to humans
Figure 2-3. Factors for weighing other data.
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1 Totality of Evidence
2 In reaching a view of the entire weight of evidence, all data and inferences are merged.
3 Figure 2-4 indicates the generalities. In fact, possible weights of evidence span a broad
4 continuum that cannot be capsulized. Most of the time the data in various lines of evidence fall in
5 the middle of the weights represented in the four figures in this section.
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Increase Weight Decrease Weight
Evidence of human causality
Data not available or do not show causality
Evidence of animal effects relevant
Data not available or not relevant
to humans
Coherent inferences
Conflicting data
Comparable metabolism and toxicokinetics
Metabolism and toxicokinetics not
between species
comparable
Mode of action comparable across species
Mode of action not comparable across species
A
Figure 2-4. Factors for weighing totality of evidence.
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The following section and the weight-of-evidence narrative discussed in Section 2.8
provide a way to state a conclusion and capture this complexity in a consistent way.
2.6.2. Descriptors for Summarizing Weight of Evidence
To express conclusions about the weight of evidence for human carcinogenic potential,
standard descriptors are utilized as part of the narrative (see Section 2.7.2.). The descriptors are
not meant to replace an explanation of the nuances of the biological evidence, but rather to
summarize it. Applying a descriptor is a matter of judgment and cannot be reduced to a formula.
Each standard descriptor may be applicable to a wide variety of potential data sets and weights of
evidence. There will always be gray areas, gradations, and borderline cases. That is why the
descriptors are presented only in the context of a weight of evidence narrative. Using them within
a narrative preserves and presents the complexity that is an essential part of the hazard
characterization. Risk managers should consider the entire range of information included in the
narrative rather than focusing simply on the descriptor.
Different conclusions may be reached for a single agent when carcinogenicity is dose or
route dependent. For instance, the agent is likely to be carcinogenic by one route of exposure but
not by others (Section 2.3.3). In this instance, more than one descriptor is used, one for each
route of exposure. Another example would be that an agent is likely carcinogenic above a certain
dose range but not likely to be carcinogenic below that range.
The descriptors are standardized and are to be used consistently from case to case. They
are part of the first sentence of the narrative. The discussions below explain descriptors which
appear in italics, and along with Appendices A and C, illustrate their use, including by route of
exposure.
"Carcinogenic To Humans"
This descriptor is appropriate when there is convincing epidemiologic evidence
demonstrating causality between human exposure and cancer.
This descriptor is also appropriate when there is an absence of conclusive epidemiologic
evidence to clearly establish a cause and effect relationship between human exposure and cancer,
but there is compelling evidence of carcinogenicity in animals and mechanistic information in
animals and humans demonstrating similar mode(s) of carcinogenic action. It is used when all of
the following conditions are met:
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There is evidence in a human population(s) of association of exposure to the
agent with cancer, but not enough to show a causal association, and
• There is extensive evidence of carcinogenicity, and
• The mode(s) of carcinogenic action and associated key events have been identified in
animals, and
• The keys events that precede the cancer response in animals have been observed in the
human population(s) that also shows evidence of an association of exposure to the
agent with cancer.
"Likely To be Carcinogenic To Humans"
This descriptor is appropriate when the available tumor effects and other key data are
adequate to demonstrate carcinogenic potential to humans. Adequate data are within a spectrum.
At one end is evidence for an association between human exposure to the agent and cancer and
strong experimental evidence of carcinogenicity in animals; at the other, with no human data, the
weight of experimental evidence shows animal carcinogenicity by a mode or modes of action that
are relevant or assumed to be relevant to humans.
"Suggestive Evidence of Carcinogenicity,
but Not Sufficient to Assess Human Carcinogenic Potential"
This descriptor is appropriate when the evidence from human or animal data is suggestive
of carcinogenicity, which raises a concern for carcinogenic effects but is judged not sufficient for a
conclusion as to human carcinogenic potential. Examples of such evidence may include: a
marginal increase in tumors that may be exposure-related, or evidence is observed only in a single
study, or the only evidence is limited to certain high background tumors in one sex of one species.
Dose-response assessment is not indicated for these agents. Further studies would be needed to
determine human carcinogenic potential.
"Data Are Inadequate for An Assessment of Human Carcinogenic Potential"
This descriptor is used when available data are judged inadequate to perform an
assessment. This includes a case when there is a lack of pertinent or useful data or when existing
evidence is conflicting, e.g., some evidence is suggestive of carcinogenic effects, but other equally
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pertinent evidence does not confirm a concern.
"Not likely To Be Carcinogenic To Humans"
This descriptor is used when the available data are considered robust for deciding that
there is no basis for human hazard concern. The judgment may be based on—
• Extensive human experience that demonstrates lack of carcinogenic effect (e.g.,
phenobarbital).
• Animal evidence that demonstrates lack of carcinogenic effect in at least two well-
designed and well-conducted studies in two appropriate animal species (in the absence
of human data suggesting a potential for cancer effects).
• Extensive experimental evidence showing that the only carcinogenic effects observed
in animals are not considered relevant to humans (e.g., showing only effects in the
male rat kidney due to accumulation of (X2u-globulin),
• Evidence that carcinogenic effects are not likely by a particular route of exposure
(Section 2.3.3.)
• Evidence that carcinogenic effects are not anticipated below a defined dose range.
2.7. TECHNICAL HAZARD CHARACTERIZATION
The hazard characterization has two functions. First, it presents results of the hazard
assessment and an explanation of how the weight-of-evidence conclusion was reached. It explains
the potential for human hazard, anticipated attributes of its expression, and mode-of-action
considerations for dose response. Second, it contains the information needed for eventual
incorporation into a risk characterization consistent with EPA guidance on risk characterization
(U.S. EPA, 1995).
The characterization summarizes the conclusions reached concerning the mode of action
of the agent and devotes particular attention to a clear statement of the strengths and weaknesses
of the inferences made and their relation to the framework for analyzing described in Chapter 2.
The implications of the mode of action for the dose-response assessment are clearly stated, along
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with the degree of confidence in those conclusions.
The characterization qualitatively describes the conditions under which the agent's effects
may be expressed in human beings. These qualitative hazard conditions are ones that are
observable in the tumor and other key data without having done either quantitative dose-response
or exposure assessment. The description includes how expression is affected by route of
exposure and dose levels and durations of exposure. Implications for disproportionate risks in
particular subpopulations, including fetuses and children, are identified when such information
exists.
The discussion of limitations of dose as a qualitative aspect of hazard addresses the
question of whether reaching a certain dose range appears to be a precondition for a hazard to be
expressed; for example, when carcinogenic effects are secondary to another toxic effect that
appears only when a certain dose level is reached. The assumption is made that an agent that
causes internal tumors by one route of exposure will be carcinogenic by another route, if it is
absorbed by the second route to give an internal dose. Conversely, if there is a route of exposure
by which the agent is not absorbed (does not cross an absorption barrier; e.g., the exchange
boundaries of skin, lung, and digestive tract through uptake processes) to any significant degree,
hazard is not anticipated by that route. An exception to the latter statement would be when the
site of contact is also the target tissue of carcinogenicity. Duration of exposure may be a
precondition for hazard if, for example, the mode of action requires cytotoxicity or a physiologic
change, or is mitogenicity, for which exposure must be sustained for a period of time before
effects occur. The characterization could note that one would not anticipate a hazard from
isolated, acute exposures. The above conditions are qualitative ones regarding preconditions for
effects, not issues of relative absorption or potency at different dose levels. The latter are dealt
with under dose-response assessment (Section 3), and their implications can only be assessed after
human exposure data are applied in the characterization of risk.
The characterization describes conclusions about mode-of-action information and its
support for recommending dose-response approaches.
The hazard characterization routinely includes the following in support of risk
characterization:
• a summary of results of the assessment;
• identification of the kinds of data available to support conclusions and explanation of
how the data fit together, highlighting the quality of the data in each line of evidence,
e.g., tumor effects, short-term studies, structure-activity relationships), and
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highlighting the coherence of inferences from the different kinds of data;
• strengths and limitations (uncertainties) of the data and assessment, including
identification of default assumptions invoked in the face of missing or inadequate data;
• identification of alternative interpretations of data that are considered equally
plausible;
• identification of any subpopulations believed to be more susceptible to the hazard than
the general population, especially attending to fetuses, infants, and children;
• conclusions about the agent's mode of action and recommended dose-response
approaches; and
• significant issues regarding interpretation of data that arose in the assessment. Typical
ones may include:
— determining causality in human studies,
— dosing (MTD), background tumor rates, relevance of animal tumors to
humans;
— weighing studies with positive and null results, considering the influence of
other available kinds of evidence; and
— drawing conclusions based on mode-of-action data versus using a default
assumption about the mode of action.
2.8. WEIGHT-OF-EVIDENCE NARRATIVE
The weight-of-evidence narrative summarizes the results of hazard assessment employing
the descriptors defined in Section 2.6.1. The narrative (about two pages in length) explains an
agent's human carcinogenic potential and the conditions of its expression. If data do not allow a
conclusion as to carcinogenicity, the narrative explains the basis of this determination. An
example narrative appears below. More examples appear in Appendix A.
The items regularly included in a narrative are:
• name of agent and Chemical Abstracts Services number, if available;
• conclusions (by route of exposure) about human carcinogenicity, using a standard
descriptor from Section 2.6.1;
• summary of human and animal tumor data on the agent or its structural analogues,
their relevance, and biological plausibility;
• other key data (e.g., structure-activity data, toxicokinetics and metabolism, short-term
studies, other relevant toxicity or clinical data);
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• discussion of possible mode(s) of action and appropriate dose-response approach(es);
and
• conditions of expression of carcinogenicity, including route, duration, and magnitude
of exposure.
Example Narrative
Aromatic Compound
CAS# XXX
CANCER HAZARD SUMMARY
Aromatic compound (AR) is carcinogenic to humans by all routes of exposure.
The weight of evidence of human carcinogenicity is based on (a) consistent evidence of
elevated leukemia incidence in studies of exposed workers and significant increases of genetic
damage in bone marrow cells and blood lymphocytes of exposed workers; (b) significantly
increased incidence of cancer in both sexes of several strains of rats and mice; (c) genetic damage
in bone marrow cells of exposed rodents and effects on intracellular signals that control cell
growth.
AR is readily absorbed by all routes of exposure and rapidly distributed throughout the
body. The mode of action of AR is not understood. A dose-response assessment that assumes
linearity of the relationship is recommended as a default.
SUPPORTING INFORMATION
Data include numerous human epidemiologic and biomonitoring studies, long-term
bioassays, and other data on effects of AR on genetic material and cell growth processes. The
key epidemiologic studies and animal studies are well conducted and reliable. The other data are
generally of good quality also.
Human Effects
Numerous epidemiologic and case studies have reported an increased incidence or a causal
relationship associating exposure to AR and leukemia. Among the studies are five for which the
design and performance as well as follow-up are considered adequate to demonstrate the causal
relationship. Biomonitoring studies of exposed workers have found dose-related increases in
chromosomal aberrations in bone marrow cells and blood lymphocytes.
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Animal Effects
AR caused increased incidence of tumors in various tissues in both sexes of several rat and
mouse strains. AR also caused chromosomal aberrations in rabbits, mice, and rats—as it does in
humans.
Other Key Data
AR itself is not DNA reactive and is not mutagenic in an array of test systems both in vitro
and in vivo. Metabolism of AR yields several metabolites that have been separately studied for
effects on carcinogenic processes. Some have mutagenic activity in test systems and some have
other effects on growth controls inside cells.
MODE OF ACTION
No rodent tumor precisely matches human leukemia in pathology. The closest parallel is a
mouse cancer of blood-forming tissue. Studies of the effects of AR at the cell level in this model
system are ongoing. As yet, the mode of action of AR is unclear, but most likely the carcinogenic
activity is associated with one or a combination of its metabolites. It is appropriate to apply a
linear approach to the dose-response assessment pending a better understanding because: (a)
genetic damage is a typical effect of AR exposure in mammals, and (b) metabolites of AR produce
mutagenic effects in addition to their other effects on cell growth controls; AR is a multitissue
carcinogen in mammals, suggesting that it is affecting a common controlling mechanism of cell
growth.
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3. DOSE-RESPONSE ASSESSMENT
Dose-response assessment evaluates potential risks to humans at exposure levels of
interest. The approach to dose-response assessment for a particular agent is based on the
conclusion reached as to its mode of action (Sections. 2.4 -2.5). The evaluation first covers the
relationship of the dose6 to the degree of response in the dose range of observation in experiments
or human studies. This evaluation is then followed by extrapolation to estimate response at lower
environmental exposure levels (ILSI, 1995). In general, three extrapolations may be made: from
high to low doses, from animal to human responses, and from one route of exposure to another.
Cancer is a disease that develops through many cell and tissue changes over time.
Traditional dose-response assessment procedures using tumor incidence as the response have
seldom taken into account the effects of key events within the whole biological process, even
though these events are the determinants of the overall dose-response. This has been due to lack
of empirical data and understanding about these events. As more data become available and our
understanding about how agents cause cancer improves, they can be used in dose-response
assessment along with the traditional procedures. These guidelines encourage use of these new
data as they become available to improve dose-response assessment.
In this discussion, "response" data include measures of key events7 considered integral to
the carcinogenic process, in addition to tumor incidence. These responses may include changes in
DNA, chromosomes, or other key macromolecules; effects on growth signal transduction,
including induction of hormonal changes; or physiological or toxic effects that affect cell
proliferation. Key events are precursors to cancer pathology; they may include proliferative
events diagnosed as precancerous, but not pathology that is judged to be cancer. Analysis of such
responses may be done along with those of tumor incidence to enhance the tumor dose-response
analysis. If dose-response analysis of non tumor key events is more informative about the
carcinogenic process for an agent, it is used in lieu of, or in conjunction with, tumor incidence
1. For this discussion, "exposure" means contact of an agent with the outer boundary of an
organism. "Applied dose" means the amount of an agent presented to an absorption barrier and
available for absorption. "Internal dose" means the amount crossing an absorption barrier (e.g.,
the exchange boundaries of skin, lung, and digestive tract) through uptake processes. "Delivered
dose" for an organ or cell means the amount available for interaction with that organ or cell (U.S.
EPA, 1992a).
2. A "key event" is an empirically observed precursor consistent with a mode of action.
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analysis for the overall dose-response assessment.
"Dose" means the "human equivalent dose" as discussed in Section 3.3, unless otherwise
noted. When animal responses are used in the assessment, the animal dose is adjusted to human
equivalence. The preferred approach for this is to use toxicokinetic modeling to compare species.
If this is not possible given the data available, a default factor for allometric scaling of oral dose is
provided. For adjustment of inhalation dose, the EPA's Reference Concentration (RfC)
methodology is used.
Coverage of the Chapter
This chapter covers: 1) consideration of mode of action in selecting dose-response
assessment approaches, 2) assessment of observed data and extrapolation procedures, 3)
analyses of response data and 4) analyses of dose data. The final section discusses dose-response
characterization.
3.1 HUMAN STUDIES
Analysis of human studies in the observed range is determined according to the type of
study and how dose and response are measured in the study. In some cases the agent may have
discernible interactive effects with another agent (e.g., asbestos and smoking), making possible
estimation of contribution of the agent and others as risk factors. Also, in some cases, estimation
of population risk in addition to, or in lieu of, individual risk may be appropriate. The following
discussions are addressed mainly to animal data. Nevertheless, if human data permit, the
principles or approaches below apply for performing dose-response assessment in two parts-
range of observation and range of extrapolation, for deriving a point of departure, and for linear
or margin of exposure analysis according to mode of action (NRC, 1999; Teta, 1999). The
approach is tailored to the nature of the human data and the mode of action data available, if any.
3.2. MODE OF ACTION AND DOSE-RESPONSE APPROACH
The cancer dose-response relationship(s) for a chemical is considered in a two step
process. First is the determination of the mode of action and dose response for each tumor type
that results in a significant increase in tumor incidence. Second is an analysis of the information
bearing on all tumor types that are increased in incidence by the chemical. The overall synthesis
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includes consideration of the number of sites, their consistency across sexes, strains and species,
the strength of the mode of action information for each tumor type, the anticipated relevance of
each tumor type to humans, and the consistency of the means of estimating risks across tumor
types.
For each tumor the mode of action and other information may support one of the
following dose response extrapolations: 1) linear, 2) nonlinear using a margin of exposure
(MOE) analysis, or 3) both linear and nonlinear (MOE) analyses. In rare cases, detailed mode of
action information may be available which allow the formulation of a biologically based model.
Examples include the following:
Factors Supporting a Linear Approach
Any of the following conclusions leads to selection of a linear dose-response assessment
approach:
• There is an absence of sufficient tumor mode of action information.
• The chemical has direct DNA mutagenic activity or other indications of DNA effects
that are consistent with linearity.
• Human exposure or body burden is high and near doses associated with key events in
the carcinogenic process (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin)
• Mode of action analysis does not support direct DNA effects, but the dose-response
relationship is expected to be linear (e.g., certain receptor-mediated effects)
Factors Supporting a Nonlinear Approach
Any of the following conclusions leads to selection of a nonlinear (margin of exposure)
approach to dose-response assessment:
• A tumor mode of action supporting nonlinearity applies (e.g., some cytotoxic and
hormonal agents such as disruptors of hormone homeostasis), and the chemical does
not demonstrate mutagenic effects consistent with linearity.
• A mode of action supporting nonlinearity has been demonstrated, and the chemical has
some indication of mutagenic activity, but it is judged not to play a significant role in
tumor causation.
Factors Supporting Both Linear And Nonlinear Approaches
Any of the following conclusions leads to selection of both a linear and nonlinear approach
to dose-response assessment. Relative support for each dose response method and advice on the
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use of that information needs to be presented. In some cases, evidence for one mode of action is
stronger that for the other, allowing emphasis to be placed on that dose-response approach. In
other cases, both modes of action are equally possible, and both dose-response approaches should
be emphasized.
• Modes of action for a single tumor type support both linear and nonlinear dose
response in different parts of the dose-response (e.g., 4,4' methylene chloride).
• A tumor mode of action supports different approaches at high and low dose; e.g., at
high dose, nonlinearity, but, at low dose, linearity (e.g., formaldehyde).
• The agent is not DNA-reactive and all plausible modes of action are consistent with
nonlinearity, but not fully established (arsenic).
• Modes of action for different tumor types support differing approaches, e.g., nonlinear
for one and linear due to lack of mode of action for the other (e.g., trichloroethylene).
The use of biologically based models is covered below.
3.3. DOSE-RESPONSE ANALYSIS
3.3.1. Modeling the Overall Process-Biologically-based Models
Generally applicable biologically-based models may be applied such as the two-stage
models of initiation plus clonal expansion and progression developed by Moolgavkar and
Knudson (1981), Chen and Farland (1991) and others. These models of the carcinogenic process
continue to be improved, but are not yet standard methods. No model of this kind is available for
standard application.
If data are extensive and sufficient to quantitatively relate specific key events in the cancer
process to neoplasia, and the purpose of the assessment is such as to justify investing the
necessary resources, a biologically-based model may be developed on an agent-specific basis.
Before developing such a model, extensive data are needed to build its form as well as to estimate
how well it conforms with the observed data to support confidence in results. Theoretical
estimates of critical parameters, such as cell proliferation rates, are not used to enable application
of such a model in the absence of data (Portier, 1987). It is possible that different models will
provide equivalent fits to the observed data but differ substantially in their projections below the
observed range. This is often the case when a model is over-parameterized (that is, there are
more parameters to be estimated than data points to be fitted), so that different combinations of
parameter estimates can yield similar results in the observed range. For this reason, critical
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parameters of a biologically based model, such as mutation and proliferation rates, are measured
in the laboratory and not estimated by curve-fitting to tumor incidence data. This approach helps
reduce model uncertainty (i.e., uncertainty due to choice of models or model structure) and
ensures that the models do not give answers that are biologically unrealistic. This approach also
provides a robustness of results (i.e., results are not likely to change substantially when fitted to a
slightly different data set), if the mode of action is sufficiently understood so that model
parameters represent rates and other quantities associated with known key events in tumor
development.
Such models are to be distinguished from toxicokinetic models (i.e., physiologically based
pharmacokinetic" models) which address dose issues, as discussed in Section 3.3.2. Effects on
dose such as saturation of metabolic pathways may introduce nonlinearities in the dose-response
relationship, but are not modes of action, and are dealt within arriving at an appropriate dose
metric.
3.3.2. Analysis in the Range of Observation
This section covers use of information about key events which may be in the context of
either human or animal data. It then discusses curve-fitting and selecting a point of departure
with regard to animal data. Last, it discusses human data.
3.3.2.1. Applying Information About Key Events
Even though a biologically-based model may not be feasible, information about key events
in the process can be used in the assessment. The principle underlying these Guidelines is to use
approaches that include as much information about these events as possible. When such
information is available, it may be used in a variety of ways:
1) If an event(s) is quantitatively described and considered key to cancer development, its
dose-response assessment in the range of observation can be used in conjunction with, or in lieu
of, the dose-response for tumor incidence to establish the point of departure for extrapolation.
[Caution must be used in using rates of molecular events such as mutation or cell proliferation or
of signal transduction. Such rates may be difficult to relate to cell or tissue changes overall. The
timing of observations of these phenomena, as well as the cell type involved, need to be linked to
other precursor events to ensure the measurement is truly a "key"event (see Section 2.5). In
many cases such rates are more appropriately used as in "2)" or "3)" below.]
2) Quantitative description of a key event(s) can be used to test whether the dose-
response for tumor incidence can be confidently extended to support a lower point of departure
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for linear extrapolation than the tumor data alone would support (e.g., to an LED01 from an
LED10).
3) Quantitative information on a key event(s) can be used to address the question of how
quickly risk decreases as dose decreases in a margin of exposure analysis.
3.3.2.2. Procedures for Analysis in the Range of Observation of Animal Studies
Curve-fitting
A curve-fitting procedure is used that is appropriate to the kind of response data in the
range of observation. This may be tumor incidence or data on a key event(s). For incidence
information, the Agency applies a standard curve-fitting procedure to provide consistency among
assessments. This procedure models incidence, adjusted for background, as an increasing function
of dose; it is available to the public on the Agency's World Wide Web site for immediate use or
for downloading (reference to be provided). The procedure identifies situations in which the
standard algorithm fails to yield a reliable point of departure, signaling the need for additional
judgment and an alternative analysis.
For tumor incidence studies that provide time-to-tumor information, more elaborate
models would be appropriate. The Agency intends to provide a time-to-tumor version of its
standard procedure in the future.
For non tumor data, curve-fitting procedures are used that are appropriate to the kind of
response data in the observed range, and are explained in each case (reference to benchmark
models to be provided).
NOAEL/LOAEL
As discussed below, the observed range of data may be represented by a
NOAEL/LOAEL procedure when a margin of exposure analysis is chosen as the default
procedure for nonlinear dose-response extrapolation.
3.3.2.3. Point of Departure for Extrapolation from Observed Animal Data
A point of departure from observed data—for tumor incidence, or for key event(s)—is
estimated to mark the beginning of extrapolation. This is a point that is either a data point or an
estimated point that can be considered to be in the range of observation, without significant
extrapolation. Depending on the kind of data available and the purpose of the analysis, there are
differing procedures for estimating the point of departure. The point of departure employs the
human equivalent dose.
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Incidence data are most amenable to curve-fitting procedures. For example, tumor data
from a rodent bioassay are traditionally modeled with curve-fitting procedures. Some key event
data may also be in the form of incidence data (e.g., hyperplasia), but more likely will be
continuous data for which currently there are not standard and consistent modeling procedures.
Continuous data include, for instance, tissue weight changes or blood levels of a hormone.
NOAEL/LOAEL procedures are available for continuous and other data as needed.
Point of Departure Using Data Suitable for Curve-fitting
When a curve-fitting procedure is applied to tumor data (see Figure 3-1) or to incidence
data on a key event, the point of departure used in most cases is the LED10—the 95% lower
confidence limit on a dose associated with 10% extra risk adjusted for background. For tumor
data, it is used as a matter of science policy to provide consistency among assessments. It is also
useful in comparing results with assessment of noncancer endpoints (U.S. EPA, 199Id). The 10%
level is selected because a 10% response is at or just below the limit of sensitivity for discerning a
statistically significant tumor increase in most long-term rodent studies (Haseman, 1983), and is
within the observed range for many other kinds of toxicity studies. Use of the lower limit takes
experimental variability and sample size into account. If a tumor incidence study has greater than
usual sensitivity and an observed response is below LED10, then a lower point for linear
extrapolation can be used to improve the assessment. [The ED10 (central estimate) is appropriate
for use in relative hazard/potency ranking among agents for priority setting because it is a more
confident comparison point among many assessments than an extrapolated point. Because of its
convenience for comparison uses, the ED10 is always presented for reference with its upper and
lower 95%) confidence limits.]
The LED10 is adopted as the standard point of departure for non tumor key event or
toxicity incidence data in order to harmonize curve-fitting procedures between cancer and
noncancer toxicity assessments. Because the NOAEL in study protocols for non tumor toxicity
can range from about a 5% to a 30% effect level (Faustman et al., 1994), adopting the 10%
effect level as the standard point of departure will accommodate most of these data sets without
departing the range of observation. The LED10 can be regarded as an improved and harmonized
estimate of the NOAEL.
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Observed Range
Extrapolation Range
Human
Exposure
of Interest
ko,
Q.
10%
0%
LED
MOE
Figure 3-1. Graphical presentation of data and extrapolation.
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Point of Departure Using Data Suitable for a NOAEL/LOAEL Procedure
The point of departure may be a NOAEL when a margin of exposure analysis is the
nonlinear dose-response approach. The kinds of data available and the circumstances of the
assessment both contribute to deciding to estimate a NOAEL or LOAEL which is not as rigorous
or as ideal as curve-fitting, but can be appropriate. The NOAEL/LOAEL procedure is used to
maintain consistency among assessments while still encouraging quantitative analyses of the data
by modeling to explore underlying phenomena.
The circumstances of an assessment can also lead to choosing a NOAEL/LOAEL
approach. If several data sets for key events and tumor response are available for an agent, and
they are a mixture of continuous and incidence data, the most practicable way to assess them
together is through a NOAEL/LOAEL approach. The purpose of the assessment also may lead to
a decision to use the NOAEL/LOAEL approach. A preliminary or screening assessment to decide
whether risk concern is high or low or to decide on additional data requirements is one example.
Similarly, the nature of the regulatory decision may be served well by this approach to assessment.
3.3.3. Analysis in the Range of Extrapolation—Default Procedures
Extrapolation from the point of departure to lower doses is usually necessary, and in the
absence of a data set rich enough to support a biologically based model, is conducted using one of
the two default procedures described below. The Agency has adopted these procedures as a
matter of science policy based on current hypotheses of the potential shapes of dose-response
curves for differing modes of action at low doses. The choice of the procedure to be used in an
individual case is a judgment based on the agent's mode of action (See Section 3.2).
3.3.3.1. Linear Procedure
For linear extrapolation, a straight line is drawn from the point of departure expressed as a
human equivalent dose (Section 3.3.2) to the origin—zero incremental dose, zero incremental
response to give a probability of extra risk. The slope of the line expresses extra risk per dose
unit (Flamm and Winbush, 1984; Gaylor and Kodell, 1980; Krewski et al., 1984). Risk is the
product of the slope and anticipated exposure. This approach to assessing risk is considered
generally conservative of public health, including sensitive subpopulations, in the absence of
specific information about the extent of human variability in sensitivity to effects. When a linear
extrapolation procedure is used, the risk characterization summary also displays the degree of
extrapolation from empirical data by showing the margin of exposure associated with exposure
scenarios of interest as below.
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J. J. 3.2. Nonlinear Extrapolation
A default assumption of nonlinearity is appropriate when there is no evidence for linearity
and sufficient evidence to support an assumption of nonlinearity. The mode of action may lead to
a dose-response relationship that is nonlinear, with response falling much more quickly than
linearly with dose, or being most influenced by individual differences in sensitivity. Alternatively,
the mode of action may theoretically have a threshold, e.g., the carcinogenicity may be a
secondary effect of toxicity or of an induced physiological change that is itself a threshold
phenomenon (see Appendix C, example 5, or Appendix D, example 2 ). The EPA does not
generally try to distinguish between modes of action that might imply a "true threshold" from
others with a nonlinear dose-response relationship. Except in unusual cases where extensive
information is available, it is not possible to distinguish between these empirically.
As a matter of science policy under this analysis, nonlinear probability functions are not
fitted to the response data to extrapolate quantitative low-dose risk estimates because different
models can lead to a very wide range of results, and there is currently no basis, generally, to
choose among them. Thus, the default procedure for nonlinear extrapolation is to conduct a
margin of exposure analysis, as described below, to evaluate concern for levels of exposure.
3.3.3.2.1. Margin of Exposure Analysis
A margin of exposure is defined as the point of departure divided by the environmental
exposure of interest. The environmental exposures of interest, for which margins of exposure are
estimated, may be actual or projected exposure levels. A risk manager decides whether a given
margin of exposure is acceptable under applicable management policy criteria. The risk
assessment provides supporting information to assist the decisionmaker in this determination.
A margin of exposure analysis is applicable if data are sufficient to presume a non-linear
dose-response function containing a significant change in slope. If, in a particular case, the
evidence indicates a biological threshold, as in the case of carcinogenicity being secondary to
another toxicity that has a threshold, an RfD8 or RfC like approach may be estimated and
considered in cancer assessment. In this case, the RfD or RfC is an estimate with uncertainty
3. A reference dose (RfD) or reference concentration (RfC) for noncancer toxicity is an estimate
with uncertainty spanning perhaps an order of magnitude of daily exposure to the human
population (including sensitive subgroups) that is anticipated to be without appreciable deleterious
effects during a lifetime. It is arrived at by dividing empirical data on effects by uncertainty
factors that consider inter- and intraspecies variability, extent of data on all important chronic
exposure toxicity endpoints, and availability of chronic as opposed to subchronic data.
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spanning perhaps an order of magnitude of daily exposure to the human population (including
sensitive subgroups) that is anticipated to be without a cancer hazard despite a lifetime of
exposure. In many cases, data may be insufficient to determine an RfD and/or an RfC for the
cancer endpoint. In that case, a margin of exposure analysis provides useful input to the decision-
maker regarding the distance between an exposure of interest and the range of observation where
cancer risk is inferred to be sub-linear.
To support a risk manager's consideration of the margin of exposure, all of the pertinent
hazard, dose-response, and human exposure information is characterized so as to provide insights
about the scientific community's current understanding of the phenomena that may be occurring
as dose (exposure) decreases substantially below the observed data. The goal is to provide as
much information as possible about the risk reduction that accompanies lowering of exposure and
the adequacy of a margin of exposure based on scientific input, recognizing that, in some cases,
legislative, sociological, and/or technological issues may also impact on the decision regarding the
acceptability of a given margin of exposure. The discussion below describes the general principles
and major elements to be considered in a margin of exposure analysis. The Agency will develop
more specific guidance on the margin of exposure approach, as recommended (SAB, 1999). The
guidance will be peer reviewed and published separately as part of the Agency's implementation
activity of these guidelines.
For a margin of exposure analysis, the point of departure would ideally be the dose where
the key events in tumor development would not occur in a heterogenous human population, thus
representing an actual "no effect level." Therefore, it is recommended that margin of exposure
analyses be based on precursor responses rather than tumor incidences, since precursor events
can often be detected with greater sensitivity( i.e. both earlier and at lower doses), providing
further input to the decision regarding acceptability of the margin of exposure. An analysis of an
actual point of departure derived from available data, however, would often contain residual
uncertainty regarding its designation as an actual no effect level for cancer in the population. The
earlier the precursor event in the carcinogenic process and the larger the margin of exposure the
more likely the exposure of interest will be without appreciable risk of cancer. To this end, some
important points to address in the analysis of the point of departure and the margin of exposure
include the following:
• Nature of the response. Is the point of departure based on tumors or on a key event
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that is a precursor to tumors? A mode of action can be represented by a sequence of
dose-response curves, where an early key event arises at a low dose, subsequent key
events at higher doses, and tumors at a still higher dose. For example, a mode of
action that begins with bladder stones and progresses through epithelial irritation and
hyperplasia before producing tumors can be represented by a sequence of dose-
response curves for stones, irritation, hyperplasia, and tumors, each curve higher on
the dose scale than its immediate precursor. A nonlinear dose-response assessment
considers more than tumors as it identifies a dose where events that can lead to tumor
development would not occur. Identification of a key event does not imply that it is
adverse in itself, only that it is an observable step preceding tumor development.
Basing a dose-response assessment on key events is intended to protect against not
only the observation of adverse effects, but also earlier damage that can lead to later
tumor development.
Thus, it is most desirable to estimate a dose-response curve for the key event precipitating
tumor development, and use this curve to estimate the point of departure. However, lack
of quantitative information on the key event may make it necessary to use tumor data
instead of key event data. In this case, the analysis of the margin of exposure must
contain an estimate of the dose-response curve for tumors plus have sufficient discussion
of the difference (on the dose scale) between no effect levels and effect levels for key
events and for tumors. A larger margin of exposure may be needed to account for
possible differences between the dose-response curves for the key events and for tumors,
and to assure decision-makers that cancer risk for the heterogeneous population
(including sensitive subgroups) is not appreciable.
• Slope of the observed dose-response curve. Have we reached a dose where tumors
or (preferably) the key precursor events would not occurl A 10-percent incidence is
typically used as a point of departure because it reflects the lowest incidence that
experimental studies can typically detect. This does not, however, mean that a 10-
percent incidence represents a level where tumors or the key precursor events would
not occur. To account for this limitation, one needs to consider the slope of the dose-
response curve, which describes how sharply the incidence declines below the point of
departure. If the dose-response curve at the point of departure is relatively steep, the
point of departure represents a point on the dose-response curve where occurrence of
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the key event(s) declines rapidly with decreasing dose. On the other hand, if the dose-
response curve is relatively shallow, then the point where the effect virtually
disappears may lie far below the point of departure. In short, the margin of exposure
needs to be larger if the analysis is based on a response(s) that has a shallow dose-
response curve compared to an analysis based on a response with a steep dose-
response curve. More guidance needs to be developed to define quantitatively what
constitutes a steep versus a shallow dose-response curve.
• Human sensitivity compared with experimental animals. How sensitive is the human
population compared with the tested animals? For this comparison, all doses should
have already been converted to equivalent human doses, using either a physiologically
based toxicokinetic model, a cross-species dosimetry model, or the default cross-
species scaling factor. These dose conversions reflect interspecies differences in
toxicokinetics, not toxicodynamics. When information is not sufficient to quantify
human sensitivity with regard to the toxicodynamics compared with the tested animals,
this uncertainty needs to be taken into account in the discussion of an adequate margin
of exposure. As with noncancer assessment, the default assumption is that the most
sensitive humans are more sensitive than the test animals. Depending on the data
available on the sensitivity of the test species to the agent and the endpoint of concern
as compared to humans, the margin of exposure decision may need to incorporate
more or less conservatism.
• Nature and extent of human variability in sensitivity. Is there information on sensitive
individuals that would be part of a heterogeneous human population? Pertinent
information would come from human studies, since animal studies, particularly those
using homogeneous animal strains, do not provide information about human
variability. When information is not sufficient to quantify the extent of human
variability in sensitivity, this uncertainty should be reflected in the discussion of an
adequate margin of exposure (also see discussion below on human exposure).
• Human Exposure. The evaluation of margin of exposure also takes into account the
expected pattern of human exposure to an agent including the magnitude, frequency,
and duration of exposure. Some modes of action involve significant duration of
exposure before tumorigenicity results. For example, stimulus of cell growth through
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hormonal or other signal disruption or as a result of damage from toxicity is reversible
if the exposure is for a short time, since homeostasis brings a return to normal levels
after cessation of exposure. Thus, for a specialized population that is occasionally and
briefly exposed to an agent with such a mode of action, an adequate of margin of
exposure would be smaller than for chronic exposure. As the duration of exposure or
frequency of exposure increases, an adequate margin of exposure would increase
accordingly.
Furthermore, if the population exposed in a particular scenario is wholly or largely
composed of a subpopulation of special concern (e.g. children) for whom evidence
indicates a special sensitivity to the agent's mode of action, an adequate margin of
exposure would be larger than for general population exposure.
To provide input regarding scientific considerations regarding the acceptability of a margin of
exposure by the risk manager, the risk assessment along with risk characterization explicitly
considers all of the hazard and dose-response and human exposure factors together. This input on
the margin of exposure is not solely a composite of individual adjustment factors to account for
missing data or knowledge gaps as discussed above. Rather, each case calls for individual
judgment, taking all of these points as a whole. It is appropriate to provide a graphical
representation of the data and dose-response modeling in the observed range, also showing
exposure levels of interest to the decision-maker (See figure 3-1.). In order to provide a frame of
reference, by way of comparison, a straight line extrapolation may be displayed to show what risk
levels would be associated with decreasing dose, if the dose-response were linear.
3.3.3.3. Linear and Nonlinear Extrapolations
Both linear and nonlinear procedures may be used in particular cases. If a mode of action
analysis finds substantial support for differing modes of action for different tumor sites, an
appropriate procedure is used for each. Both procedures may also be appropriate to discuss
implications of complex dose-response relationships. For example, if it is apparent that an agent
is both DNA reactive and is highly active as a promotor at high doses, and there are insufficient
data for modeling, both linear and nonlinear default procedures may be needed to decouple and
consider the contribution of both phenomena.
3.3.3.4. Use of Toxicity Equivalence Factors and Relative Potency Estimates
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A toxicity equivalence factor (TEF) procedure is one used to derive quantitative dose-
response estimates for agents that are members of a category or class of agents. TEFs are based
on shared characteristics that can be used to order the class members by carcinogenic potency
when cancer bioassay data are inadequate for this purpose (U.S. EPA, 1991c). The ordering is by
reference to the characteristics and potency of a well-studied member or members of the class.
Other class members are indexed to the reference agent(s) by one or more shared characteristics
to generate their TEFs. The TEFs are usually indexed at increments of a factor of 10. Very good
data may permit a smaller increment to be used. Shared characteristics that may be used are, for
example, receptor-binding characteristics, results of assays of biological activity related to
carcinogenicity, or structure-activity relationships.
TEFs are generated and used for the limited purpose of assessment of agents or mixtures
of agents in environmental media when better data are not available. When better data become
available for an agent, its TEF should be replaced or revised. Criteria for constructing TEFs are
given in U.S. EPA (1991b). The criteria call for data that are adequate to support summing doses
of the agents in mixtures. To date, adequate data to support use of TEF's has been found in only
one class of compounds (dioxins) (U.S. EPA, 1989a).
Relative potencies can be similarly derived and used for agents with carcinogenicity or
other supporting data. These are conceptually similar to TEFs, but they are less firmly based in
science and do not have the same level of data to support them. They are used only when there is
no better alternative.
The uncertainties associated with both TEFs and relative potencies are explained
whenever they are used.
3.4. RESPONSE DATA
Response data for analysis include tumor incidence data from human or animal studies as
well as data on other responses as they relate to an agent's carcinogenicity, such as effects on
growth control processes or cell macromolecules or other toxic effects. Tumor incidence data are
ordinarily the basis of dose-response assessment, but other response data can augment such
assessment or provide separate assessments of carcinogenicity or other important effects.
Data on carcinogenic processes underlying tumor effects may be used to support
biologically based or case-specific models. Other options for such data exist. If confidence is
high in the linkage of a precursor effect and the tumor effect, the assessment of tumor incidence
may be extended to lower dose levels by linking it to the assessment of the precursor effect
(Swenberg et al., 1987). Even if a quantitative link is not appropriate, the assessment for a
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precursor effect may provide a view of the likely shape of the dose-response curve for tumor
incidence below the range of tumor observation (Cohen and Ellwein, 1990; Choy, 1993). If
responses other than tumor incidence are regarded as better representations of the carcinogenicity
of the agent, they may be used in lieu of tumor responses. For example, if it is concluded that the
carcinogenic effect is secondary to another toxic effect, the dose-response for the other effect will
likely be more pertinent for risk assessment. As another example, if disruption of hormone
activity is the key mode of action of an agent, data on hormone activity may be used in lieu of
tumor incidence data.
If adequate positive human epidemiologic response data are available, they provide an
advantageous basis for analysis since concerns about interspecies extrapolation do not arise.
Adequacy of human exposure data for quantification is an important consideration in deciding
whether epidemiologic data are the best basis for analysis in a particular case. If adequate
exposure data exist in a well-designed and well-conducted epidemiologic study that detects no
effects, it may be possible to obtain an upper-bound estimate of the potential human risk to
provide a check on plausibility of available estimates based on animal tumor or other responses,
e.g., do confidence limits on one overlap the point estimate of the other?
When animal studies are used, response data from a species that responds most like
humans should be used if information to this effect exists. If this is unknown and an agent has
been tested in several experiments involving different animal species, strains, and sexes at several
doses and different routes of exposure, all of the data sets are considered and compared, and a
judgment is made as to the data to be used to best represent the observed data and important
biological features such as mode of action. Appropriate options for presenting results include:
• use of a single data set,
• combining data from different experiments (Stiteler et al., 1993; Vater et al., 1993),
• showing a range of results from more than one data set,
• showing results from analysis of more than one statistically significant tumor response
based on differing modes of action,
• representing total response in a single experiment by combining animals with
statistically significant tumors at more than one site, or
• a combination of these options.
The approach judged to best represent the data is presented with the rationale for the judgment,
including the biological and statistical considerations involved. The following are some points to
consider:
• quality of study protocol and execution,
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• proportion of malignant neoplasms,
• latency of onset of neoplasia,
• number of data points to define the relationship of dose and response,
• background incidence in test animal,
• differences in range of response among species, sexes, strains,
• most sensitive responding species, and
• availability of data on related precursor events to tumor development.
Analyses of carcinogenic effects other than tumor incidence are similarly presented and evaluated
for their contribution to a best judgment on how to represent the biological data for dose-
response assessment.
3.5. DOSE DATA
Whether animal experiments or epidemiologic studies are the sources of data, questions
need to be addressed in arriving at an appropriate measure of dose for the anticipated
environmental exposure. Among these are:
• whether the dose is expressed as an environmental concentration, applied dose, or
delivered dose to the target organ,
• whether the dose is expressed in terms of a parent compound, one or more
metabolites, or both,
• the impact of dose patterns and timing where significant,
• conversion from animal to human doses, where animal data are used, and
• the conversion metric between routes of exposure where necessary and appropriate.
In practice, there may be little or no information on the concentration or identity of the active
form at a target; being able to compare the applied and delivered doses between routes and
species is the ideal, but is rarely attained. Even so, the objective is to use available data to obtain
as close to a measure of internal or delivered dose as possible.
The following discussion assumes that the analyst will have data of varying detail in
different cases about toxicokinetics and metabolism. Discussed below are approaches to basic
data that are most frequently available, as well as approaches and judgments for improving the
analysis based on additional data. The estimation of dose in human studies is tailored to the form
of dose data available.
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3.5.1. Interspecies Adjustment of Dose—Adult Human
When adequate data are available, the doses used in animal studies can be adjusted to
equivalent human doses using toxicokinetic information on the particular agent. The methods
used should be tailored to the nature of the data on a case-by-case basis. In rare cases, it may also
be possible to make adjustments based on toxicodynamic considerations. In most cases, however,
there are insufficient data available to compare dose between species. In these cases, the estimate
of human equivalent dose is based on science policy default assumptions. The defaults described
below are modified or replaced whenever better comparative data on toxicokinetic or metabolic
relationships are available. The availability and discussion of the latter also may permit reduction
or discussion of uncertainty in the analysis.
For oral exposure, the default assumption is that delivered doses are related to applied
dose by a power of body weight. This assumption rests on the similarities of mammalian
anatomy, physiology, and biochemistry generally observed across species. This assumption is
more appropriate at low applied dose concentrations where sources of nonlinearity, such as
saturation or induction of enzyme activity, are less likely to occur. To derive an equivalent human
oral dose from animal data, the default procedure is to scale daily applied doses experienced for a
lifetime in proportion to body weight raised to the 0.75 power (W0 75). Equating exposure
concentrations in parts per million units for food or water is an alternative version of the same
default procedure because daily intakes of these are in proportion to W°75. The rationale for this
factor rests on the empirical observation that rates of physiological processes consistently tend to
maintain proportionality with W°75. A more extensive discussion of the rationale and data
supporting the Agency's adoption of this scaling factor is in U.S. EPA, 1992b. Information such
as blood levels or exposure biomarkers or other data that are available for interspecies comparison
are used to improve the analysis when possible.
The default procedure to derive an human equivalent concentration of inhaled particles
and gases is described in U.S. EPA (1994) and Jarabek (1995a,b). The methodology estimates
respiratory deposition of inhaled particles and gases and provides methods for estimating internal
doses of gases with different absorption characteristics. The method is able to incorporate
additional toxicokinetics and metabolism to improve the analysis if such data are available.
3.5.2. Adjustment of Dose from Adults to Children
Slope factors and unit risk estimates for lifetime exposure incorporate exposure factors
that are based on adults (specifically, body weight, breathing rate, and drinking water ingestion
rate). When these unit risk estimates are used to assess risks from less-than-lifetime exposure that
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occurs during childhood, adjustments for differences between adults and children may be
appropriate.
Inhalation unit risk estimates: Section 3.5.1 specifies that the inhalation methodology
(U.S. EPA, 1994) be used for inhaled concentrations when agent-specific data are insufficient to
develop a case-specific dosimetry model. The methodology incorporates exposure factors based
on a 70-kg adult who breathes at a plausibly high rate of 20 m3/d. Because children breathe more
air per unit of body weight (U.S. EPA, 1998), use of adult exposure factors may not be
appropriate. Consequently, inhalation unit risk estimates are adjusted to reflect a child's body
weight and breathing rate. For example, the following calculation adjusts an (adult) unit risk
estimate of lxl0"4 per ug/m3 so that it applies to a 9-kg infant who breathes 4.5 m3/d:
(lxlO"4 per ug/m3) x (4.5 m3/d / 20 m3/d) / (9 kg / 70 kg) = 1.75xl0"4 per ug/m3.
For inhaled gases and aerosols, this adjustment is intended to provide the same degree of
health-conservatism for children and adults. For inhaled particles, the adjustment does not take
into account the different size and spacing of airways of children and adults; this difference could
result in children and adults retaining particles with a different size distribution and different
toxicologic properties. To reduce this uncertainty, EPA is developing a default dosimetry model
for children that is based on children's inhalation parameters.
Drinking water unit risk estimates: Similarly, drinking water unit risk estimates
incorporate exposure factors based on a 70-kg adult who drinks water at a plausibly high rate of
2 L/d. Because children drink more water per unit of body weight (U.S. EPA, 1997c), use of
adult exposure factors may not be appropriate. Consequently, drinking water unit risk estimates
will be adjusted to reflect a child's body weight and drinking water ingestion rate.
Oral slope factors: Oral slope factors incorporate a cross-species scaling factor based on
equivalence of mg/kg3/4-d (U.S. EPA, 1992b). This cross-species factor is intended to achieve
equivalence in lifetime cancer risk in different mammalian species. When risks from childhood
exposure are being assessed, the child's weight is not substituted for an adult weight in the cross-
species scaling factor. There are several reasons why using the child's weight in the cross-species
factor may not be appropriate:
• Using the child's weight instead of an adult weight assumes that children have faster
metabolism, leading to faster clearance, smaller body burdens, and smaller risks.
Although children generally metabolize and eliminate many chemicals faster than
adults, this is not true in all cases (Renwick, 1998).
• The data supporting the 3/4-power factor pertain to cross-species equivalence, a
fundamentally different question from determining equivalence across different life
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stages of a single species.
• Although exposure may begin during childhood, subsequent events that complete the
carcinogenesis process may continue into adulthood.
Using an adult body weight is also a science policy choice that provides some degree of
health-conservatism for children in view of the uncertainties in extrapolating risks to children.
Quantitatively, the effect of this choice is rather modest; for example, basing the scaling factor on
a 70-kg adult instead of a 10-kg child results in risk estimates that are 1.6 times higher
([70/10][1"3/4] = 1.6).
Dermal exposure: The risk of distal-site cancers from the fraction of a dermal exposure
that is systemically absorbed is sometimes assessed by reducing the oral slope factor by a dermal
absorption factor that reflects the ratio of absorption by the dermal route to absorption by the oral
route. Use of a dermal absorption factor based on adults could increase the uncertainty in a risk
assessment of childhood exposure. Neonates, especially premature infants, have much greater
skin absorption than older children or adults (Schilter et al., 1996).
The risk of skin cancer from dermal exposure, in particular, from the fraction that remains
on the skin and is not systemically absorbed, has generally not been addressed because methods to
do so have not been developed. In order to assess children's risks from this important pathway,
methodological research is needed in this area.
3.5.3. Toxicokinetic Analyses
Physiologically based mathematical models are potentially the most comprehensive way to
account for toxicokinetic processes affecting dose. Models build on physiological compartmental
modeling and attempt to incorporate the dynamics of tissue perfusion and the kinetics of enzymes
involved in metabolism of an administered compound.
A comprehensive model requires the availability of empirical data on the carcinogenic
activity contributed by parent compound and metabolite or metabolites and data by which to
compare kinetics of metabolism and elimination between species. A discussion of issues of
confidence accompanies presentation of model results (Monro, 1992). This includes
considerations of model validation and sensitivity analysis that stress the predictive performance
of the model. When a delivered dose measure is used in animal to human extrapolation of dose-
response data, the assessment should discuss the confidence in the assumption that the
toxicodynamics of the target tissue(s) will be the same in both species. Toxicokinetic data can
improve dose-response assessment by accounting for sources of change in proportionality of
applied to internal or delivered dose at various levels of applied dose. Many of the sources of
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potential nonlinearity involve saturation or induction of enzymatic processes at high doses. An
analysis that accounts for nonlinearity (for instance, due to enzyme saturation kinetics) can assist
in avoiding overestimation or underestimation of low dose-response otherwise resulting from
extrapolation from a sublinear or supralinear part of the experimental dose-response curve
(Gillette, 1983). Toxicokinetic processes tend to become linear at low doses, an expectation that
is more robust than low-dose linearity of response (Hattis, 1990). Accounting for toxicokinetic
nonlinearities allows better description of the shape of the curve at relatively high levels of dose in
the range of observation, but cannot determine linearity or nonlinearity of response at low dose
levels (Lutz, 1990a; Swenberg et al., 1987).
Toxicokinetic modeling results may be presented as the preferred method of estimating
human equivalent dose or in parallel discussion with default assumptions depending on relative
confidence in the modeling.
3.5.4. Route-to-Route Extrapolation
Judgments frequently need to be made about the carcinogenicity of an agent through a
route of exposure different than the one in the underlying studies. For example, exposures of
interest may be through inhalation of an agent tested primarily through animal feeding studies or
through ingestion of an agent that showed positive results in human occupational studies from
inhalation exposure.
Route-to-route extrapolation has both qualitative and quantitative aspects. For the
qualitative aspect, the assessor weighs the degree to which positive results through one route of
exposure in human or animal studies support a judgment that similar results would have been
observed in appropriate studies using the route of exposure of interest. In general, confidence in
making such a judgment is strengthened when the tumor effects are observed at a site distant from
the portal of entry and when absorption through the route of exposure of interest is similar to
absorption via the tested routes. In the absence of contrary data, the qualitative default
assumption is that, if the agent is absorbed by a route to give an internal dose, it may be
carcinogenic by that route. (See section 2.7.1.)
When a qualitative extrapolation can be supported, quantitative extrapolation may still be
problematic in the absence of adequate data. The differences in biological processes among
routes of exposure (oral, inhalation, dermal) can be great because of, for example, first-pass
effects and differing results from different exposure patterns. There is no generally applicable
method for accounting for these differences in uptake processes in quantitative route-to-route
extrapolation of dose-response data in the absence of good data on the agent of interest.
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Therefore, route-to-route extrapolation of dose data relies on a case-by-case analysis of available
data. When good data on the agent itself are limited, an extrapolation analysis can be based on
expectations from physical and chemical properties of the agent, properties and route-specific
data on structurally analogous compounds, or in vitro or in vivo uptake data on the agent. Route-
to-route uptake models may be applied if model parameters are suitable for the compound of
interest. Such models are currently considered interim methods; further model development and
validation is awaiting the development of more extensive data (see generally, Gerrity and Henry,
1990). For screening or hazard ranking, route-to-route extrapolation may be based on assumed
quantitative comparability as a default, as long as it is reasonable to assume absorption by
compared routes. When route-to-route extrapolation is used, the assessor's degree of confidence
in both the qualitative and quantitative extrapolation should be discussed in the assessment and
highlighted in the dose-response characterization.
3.5.5. Dose Averaging
The cumulative dose received over a lifetime, expressed as lifetime average daily dose, is
generally considered an appropriate default measure of exposure to a carcinogen (Monro, 1992).
The assumption is made that a high dose of a carcinogen received over a short period of time is
equivalent to a corresponding low dose spread over a lifetime. While this is a reasonable default
assumption based on theoretical considerations, departures from it are expected. Another
approach is needed in some cases, such as when dose-rate effects are noted (e.g., formaldehyde).
Cumulative dose may be replaced, as appropriate and justified by the data, with other dose
measures. In such cases, modifications to the default assumption are made to take account of
these effects; the rationale for the selected approach is explained.
In cases where a mode of action or other feature of the biology has been identified that has
special dose implications for sensitive subpopulations (e.g., differential effects by sex or
disproportionate impacts of early-life exposure), these are explained and are recorded to guide
exposure assessment and risk characterization. Special problems arise when the human exposure
situation of concern suggests exposure regimens (e.g., route and dosing schedule) that are
substantially different from those used in the relevant animal studies. These issues are explored
and pointed out for attention in the exposure assessment and risk characterization.
3.6. DISCUSSION OF UNCERTAINTIES
The exploration of significant uncertainties in data for dose and response and in
extrapolation procedures is part of the assessment. The presentation distinguishes between model
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uncertainty and parameter uncertainty. Model uncertainty is an uncertainty about a basic
biological question. For example, a default, linear dose-response extrapolation may have been
made based on tumor and other key evidence supporting the view that the model for an agent's
mode of action is a DNA-reactive process. Discussion of the confidence in the extrapolation is
appropriately done qualitatively or by showing results for alternatives that are equally plausible. It
is not useful, for example, to conduct quantitative uncertainty analysis running multiple forms of
linear models. This would obviate the function of the policy default.
Parameter uncertainties deal with numbers representing statistical or analytical measures
of variance or error in data or estimates. Uncertainties in parameters are described quantitatively,
if practicable, through sensitivity analysis and statistical uncertainty analysis. With the recent
expansion of readily available computing capacity, computer methods are being adapted to create
simulated biological data that are comparable with observed information. These simulations can
be used for sensitivity analysis, for example, to analyze how small, plausible variations in the
observed data could affect dose-response estimates. These simulations can also provide
information about experimental uncertainty in dose-response estimates, including a distribution of
estimates that are compatible with the observed data. Because these simulations are based on the
observed data, they cannot assist in evaluating the extent to which the observed data as a whole
are idiosyncratic rather than typical of the true situation. If quantitative analysis is not possible,
significant parameter uncertainties are described qualitatively. In either case, the discussion
highlights uncertainties that are specific to the agent being assessed, as distinct from those that are
generic to most assessments.
Estimation of the applied dose in a human study has numerous uncertainties such as the
exposure fluctuations that humans experience compared with the controlled exposures received
by animals on test. In a prospective cohort study, there is opportunity to monitor exposure and
human activity patterns for a period of time that supports estimation of applied dose (U.S. EPA,
1992a). In a retrospective study, exposure may be based on monitoring data but is often based on
human activity patterns and levels reconstructed from historical data, contemporary data, or a
combination of the two. Such reconstruction is accompanied by analysis of uncertainties
considered with sensitivity analysis in the estimation of dose (Wyzga, 1988; U.S. EPA, 1986a).
These uncertainties can also be assessed for any confounding factor for which a quantitative
adjustment of dose-response data is made (U.S. EPA, 1984).
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3.7. TECHNICAL Dose-response CHARACTERIZATION
As with hazard characterization, the dose-response characterization serves the dual
purposes of presenting a technical characterization of the assessment results and supporting the
risk characterization.
The characterization presents the results of analyses of dose data, of response data, and of
dose-response. When alternative approaches are plausible and persuasive in selecting dose data,
response data, or extrapolation procedures, the characterization follows the alternative paths of
analysis and presents the results. The discussion covers the question of whether any should be
preferred over others because it (or they) better represents the available data or corresponds to
the view of the mechanism of action developed in the hazard assessment. The results for different
tumor types by sex and species are provided along with the one(s) preferred. Similarly, results for
responses other than tumor incidence are shown if appropriate.
Numerical dose-response estimates are presented to one significant figure to prevent an
inappropriate sense of high precision. However, since rounding can introduce significant errors in
a calculation, the rounding should be performed explicitly in the presentation of results; the actual
calculations are not done with intermediate rounding. Numbers are qualified as to whether they
represent central tendency or upper bounds and whether the method used is inherently more likely
to overestimate or underestimate (Krewski et al., 1984).
In cases where a mode of action or other feature of the biology has been identified that has
special implications for early-life exposure, differential effects by sex, or other concerns for
sensitive subpopulations, these are explained. Similarly, any expectations that high dose-rate
exposures may alter the risk picture for some portion of the population are described. These and
other perspectives are recorded to guide exposure assessment and risk characterization. Whether
the lifetime average daily dose or another measure of dose should be considered for differing
exposure scenarios is discussed.
Uncertainty analyses, qualitative or quantitative if possible, are highlighted in the
characterization.
The dose-response characterization routinely includes the following, as appropriate for the
data available:
• identification of the kinds of data available for analysis of dose and response and for
dose-response assessment,
• results of assessment as above,
• explanation of analyses in terms of quality of data available,
• selection of study/response and dose metric for assessment,
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• discussion of implications of variability in human susceptibility, including for
susceptible subpopulation,
• applicability of results to varying exposure scenarios—issues of route of exposure, dose
rate, frequency, and duration,
• discussion of strengths and limitations (uncertainties) of the data and analyses that are
quantitative as well as qualitative, and
• special issues of interpretation of data, such as:
selecting dose data, response data, and dose-response approach(es),
use of meta-analysis,
uncertainty and quantitative uncertainty analysis.
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4. TECHNICAL EXPOSURE CHARACTERIZATION
Exposure assessment is the determination (qualitative and quantitative) of the magnitude,
frequency, and duration of exposure (EPA, 1992). The following section provides a brief
overview of exposure assessment principles with an emphasis on issues related to carcinogenic
risk assessment. The information presented here should be used in conjunction with other
guidances including: the 1992 Guidelines for Exposure Assessment, the 1995 Policy and
Guidance for Risk Characterization, the 1997 Exposure Factors Handbook, the 1997 Policy for
Use of Probabilistic Analysis in Risk Assessments, and the 1997 Guiding Principles for Monte
Carlo Analysis. In addition, program specific guidelines for exposure assessment should be
consulted.
Exposure assessment generally consists of four major steps: defining the assessment
questions, selecting or developing the conceptual and mathematical models, collecting data or
selecting and evaluating available data, and exposure characterization. Each of these steps is
briefly described below.
Defining the Assessment Questions
In providing a clear and unambiguous statement of the purpose and scope of the exposure
assessment (EPA, 1997a), consider the following.
~ The management objectives of the assessment will determine whether deterministic
screening level analyses are adequate or whether full probabilistic exposure
characterization is needed.
~ Identify and include all important sources (e.g., pesticide applications), pathways (e.g.,
food or water), and routes (e.g., ingestion, inhalation, and dermal) of exposure in the
assessment. If a particular source, pathway, or route is omitted, a clear and
transparent explanation should be provided.
~ Separate analyses should be conducted for each definable subgroup within the
population of interest. In particular, subgroups that are believed to be highly exposed
or susceptible to a particular health effect should be studied. This includes people with
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certain diseases or genetic susceptibilities, and others whose behavior or physiology
may lead to higher exposure or susceptibility. Consider the following examples.
~ Physiological differences between men and women (e.g., body weight and
inhalation rate) may lead to important differences in exposures. See, for example,
the discussion in the Exposure Factors Handbook, Appendix 1A (EPA, 1997c).
~ Pregnant and lactating women may have exposures that differ from the general
population (e.g., slightly higher water consumption) (EPA, 1997c). Further,
exposure to pregnant women may result in exposure to the developing fetus.
(NAS, 1993).
~ Children consume more food per body weight than adults while consuming fewer
types of foods (ILSI, 1992, NAS, 1993 and EPA, 1997c). In addition, children
engage in crawling and mouthing (i.e., putting hands and objects in the mouth)
behaviors which can increase their exposures.
~ The elderly and disabled may have important differences in their exposures due to
a more sedentary lifestyle (EPA, 1997c). In addition, the health status of this
group may affect their susceptibility to the detrimental effects of exposure.
For further guidance, see the Guidelines for Exposure Assessment, § 3 (EPA, 1992).
Selecting or Developing the Conceptual and Mathematical Models
Carcinogen risk assessment models are generally based on the premise that risk is
proportional to total lifetime dose. Therefore, the exposure metric used for carcinogenic risk
assessment is the Lifetime Average Daily Dose (LADD). The LADD is typically used in
conjunction with the Cancer Slope Factor (CSF) to calculate individual excess cancer risk. It is
an estimate of the daily intake of a carcinogenic agent throughout the entire life of an individual.
Depending on the objectives of the assessment, the LADD may be calculated deterministically
(using point estimates for each factor to derive a point estimate of the exposure) or stochastically
(using probability distributions to represent each factor and such techniques as Monte Carlo
analysis to derive a distribution of the LADD) (EPA, 1997b). Stochastic analyses may help to
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identify certain population segments that are highly exposed and may need to be assessed as a
special subgroup. For further guidance, see the Guidelines for Exposure Assessment, § 5.3.5.2
(EPA, 1992).
When the route of exposure is inhalation or dermal contact, derivation of the LADD will
often require an approach to "route-to-route extrapolation." The CSF and other measures of
toxicity are typically derived from oral administered doses in animal studies. Therefore, for
ingestion exposures in a human population it is not usually necessary to make adjustments to
account for route specific differences in absorption and uptake. However, for inhalation and
dermal exposures, such adjustments may be necessary. For further guidance, see the Guidelines
for Exposure Assessment, § 2.1.4 (EPA, 1992).
As discussed elsewhere in these guidelines, there may be cases where the mode of action
indicates that dose rates are important in the carcinogenic process. In these cases, short term,
less-than-lifetime exposure estimates may be more appropriate for risk assessment than the
LADD. Such estimates could be used to calculate the margin (MOE) that exists between
exposure and the point of departure derived in the dose-response assessment.
Collecting Data or Selecting and Evaluating Available Data
After the assessment questions have been defined and the conceptual and mathematical
models have been developed, it is necessary to compile and evaluate existing data or, if necessary,
to collect new data. Depending on the exposure scenario under consideration, data on a wide
variety of exposure factors may be needed. The U.S. EPA Exposure Factors Handbook (EPA,
1997c) contains a large compilation of exposure data with some analysis and recommendations.
Some of these data are organized by age groups to assist with assessing such subgroups as
children. See, for example, the Exposure Factors Handbook, Volume 1, Chapter 3 (EPA, 1997c).
When using these existing data, it is important to evaluate the quality of the data and the extent to
which the data are representative of the population under consideration. The U.S. EPA Guidance
for Data Quality Assessment (EPA, 1996) and program specific guidances can provide further
assistance for evaluating existing data.
When existing data fail to provide an adequate surrogate for the needs of a particular
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assessment, it will be necessary to collect new data. Such data collection efforts should be guided
by the references listed above (e.g., the Guidance for Data Quality Assessment and program
specific guidance). Once again, subgroups of concern are an important consideration in any data
collection effort.
Exposure Characterization
The exposure characterization is a technical characterization that presents the assessment
results and supports the risk characterization. It provides a statement of the purpose, scope, and
approach used in the assessment, identifying the exposure scenarios and population subgroups
covered. It provides estimates of the magnitude, frequency, duration, and distribution of
exposures among members of the exposed population as the data permit. It identifies and
compares the contribution of different sources, pathways, and routes of exposure. In particular, a
qualitative discussion of the strengths and limitations (uncertainties) of the data and models are
presented.
The discussion of uncertainties is a critical component of the exposure characterization.
Uncertainties can arise out of problems with the conceptual and mathematical models.
Uncertainties can also arise from poor data quality and data that are not quite representative of
the population or scenario of interest. Consider the following examples of uncertainties.
~ National data (i.e., data collected to represent the entire U.S. population) may not be
representative of exposures occurring within a regional or local population.
~ Use of short term data to infer chronic, lifetime exposures must be done with caution.
Using short term data to estimate long term exposures has the tendency to
underestimate the number of people exposed, while overestimating the exposure levels
experienced by those in the upper end (i.e., above the 90th percentile) of the exposure
distribution. For further guidance, refer to the Guidelines for Exposure Assessment, §
5.3.1 (EPA, 1992).
~ Children's behavior may lead to relatively high but intermittent exposures (EPA,
1998). This pattern of exposure, "one that gradually declines over the developmental
period and which remains relatively constant thereafter" is not accounted for in the
LADD model (ILSI, 1992). Further the physiological characteristics of children may
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lead to important differences in exposure. Some of these differences can be accounted
for in the LADD model. For further guidance, see the Guidelines for Exposure
Assessment, § 5.3.5.2 (EPA, 1992).
4 Overall, the exposure characterization should provide a full description of the sources,
5 pathways, and routes of exposure. The characterization also should include a full description of
6 the populations assessed. In particular highly exposed or susceptible subgroups should be
7 discussed. For further guidance on the exposure characterization, consult the 1992 Guidelines for
8 Exposure Assessment (EPA, 1992), the 1995 Policy and Guidance for Risk Characterization
9 (EPA, 1995b and a) and EPA's Rule Writer's Guide to Executive Order 13045 (especially
10 Attachment C: Technical Support for Risk Assessors—Suggestions for Characterizing Risks to
11 Children) (EPA, 1999).
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5. RISK CHARACTERIZATION
5.1. PURPOSE
EPA has developed general guidance on risk characterization for use in all of its risk
assessment activities. Administrator Carol Browner has issued a policy statement on risk
characterization, the core of which is the following mandate:
Each risk assessment prepared in support of decision making at EPA
should include a risk characterization that follows the principles and reflects the
values outlined in this policy. A risk characterization should be prepared in a
manner that is clear, transparent, reasonable, and consistent with other risk
characterizations of similar scope prepared across programs in the Agency.
Further, discussion of risk in all EPA reports, presentations, decision packages,
and other documents should be substantively consistent with the risk
characterization. The nature of the risk characterization will depend upon the
information available, the regulatory application of the risk information, and the
resources (including time) available. In all cases, however, the assessment
should identify and discuss all the major issues associated with determining the
nature and extent of the risk and provide commentary on any constraints
limiting fuller exposition. (U.S. EPA, 1995)
EPA is also developing a Risk Characterization Handbook (draft available as publication number
EPA/600/R-99/025, dated March 1999), which provides detailed guidance to Agency staff. The
discussion below does not attempt to duplicate this material but summarizes its applicability to
carcinogen risk assessment.
The risk characterization process includes an integrative analysis of the major results of
the risk assessment which is summarized for the risk manager in a nontechnical discussion that
minimizes the use of technical terms. It is an appraisal of the science that informs the risk
manager in his/her public health decisions, as do other decision-making analyses of economic,
social, or technology issues. It also serves the needs of other interested readers. The summary is
an information resource for preparation of risk communication information, but being somewhat
technical, is not itself the usual vehicle for communication with every audience.
The integrative analysis brings together the assessments of hazard, dose response, and
exposure to make risk estimates for the exposure scenarios of interest. This analysis is generally
much more extensive than the Risk Characterization Summary. It may be peer-reviewed or
subject to public comment along with the summary in preparation for an Agency decision. The
integrative analysis may be titled differently by different EPA programs (e.g., "Staff Paper" for
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criteria air pollutants), but it typically will identify exposure scenarios of interest in decision
making and present risk analyses associated with them. Some of the analyses may concern
scenarios in several media; others may examine, for example, only drinking water risks. The
integrative analysis also may be the document that contains quantitative analyses of uncertainty.
The values supported by a risk characterization throughout the process are transparency
in environmental decision making, clarity in communication, consistency in core assumptions and
science policies from case to case, and reasonableness. While it is appropriate to err on the side
of protection of health and the environment in the face of scientific uncertainty, common sense
and reasonable application of assumptions and policies are essential to avoid unrealistic estimates
of risk (U.S. EPA, 1995). Both integrative analyses and the Risk Characterization Summary
present an integrated and balanced picture of the analysis of the hazard, dose response, and
exposure. The risk analyst should provide summaries of the evidence and results and describe the
quality of available data and the degree of confidence to be placed in the risk estimates.
Important features include the constraints of available data and the state of knowledge, significant
scientific issues, and significant science and science policy choices that were made when
alternative interpretations of data existed (U.S. EPA, 1995). Choices made about using default
assumptions or data in the assessment are explicitly discussed in the course of analysis, and if a
choice is a significant issue, it is highlighted in the summary.
5.2. APPLICATION
Risk characterization is a necessary part of generating any Agency report on risk, whether
the report is preliminary, to support allocation of resources toward further study, or
comprehensive, to support regulatory decisions. In the former case, the detail and sophistication
of the characterization are appropriately small in scale; in the latter case, appropriately extensive.
Even if a document covers only parts of a risk assessment (hazard and dose-response analyses, for
instance), the results of these are characterized.
Risk assessment is an iterative process that grows in depth and scope in stages from
screening for priority making, to preliminary estimation, to fuller examination in support of
complex regulatory decision making. Default assumptions are used at every stage because no
database is ever complete, but they are predominant at screening stages and are used less as more
data are gathered and incorporated at later stages. Various provisions in EPA-administered
statutes require decisions based on findings that represent all stages of iteration. There are close
to 30 provisions within the major statutes that require decisions based on risk, hazard, or
exposure assessment. For example, Agency review of pre-manufacture notices under Section 5 of
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the Toxic Substances Control Act relies on screening analyses, while requirements for industry
testing under Section 4 of that act rely on preliminary analyses of risk or simply of exposure. At
the other extreme, air quality criteria under the Clean Air Act rest on a rich data collection
required by statute to undergo reassessment every few years. There are provisions that require
ranking of hazards of numerous pollutants—by its nature a screening level of analysis—and other
provisions that require a full assessment of risk. Given this range in the scope and depth of
analyses, not all risk characterizations can or should be equal in coverage or depth. The risk
assessor must carefully decide which issues in a particular assessment are important to present,
choosing those that are noteworthy in their impact on results. For example, health effect
assessments typically rely on animal data since human data are rarely available. The objective of
characterization of the use of animal data is not to recount generic issues about interpreting and
using animal data. Agency guidance documents cover these. Instead, the objective is to call out
any significant issues that arose within the particular assessment being characterized and inform
the reader about significant uncertainties that affect conclusions.
5.3. PRESENTATION OF RISK CHARACTERIZATION SUMMARY
The presentation is a nontechnical discussion of important conclusions, issues, and
uncertainties that uses the hazard, dose-response, exposure, and integrative analyses for technical
support. The primary technical supports within the risk assessment are the hazard
characterization, dose-response characterization, and exposure characterization described in this
guideline. The risk characterization is derived from these. The presentation should fulfill the aims
outlined in the purpose section above.
5.4. CONTENT OF RISK CHARACTERIZATION SUMMARY
Specific guidance on hazard, dose response, and exposure characterization appears in
previous sections. Overall, the risk characterization routinely includes the following, capturing
the important items covered in hazard, dose response, and exposure characterization:
• primary conclusions about hazard, dose response, and exposure, including equally
plausible alternatives;
• nature of key supporting information and analytic methods;
• risk estimates and their attendant uncertainties, including key uses of default
assumptions when data are missing or uncertain;
• statement of the extent of extrapolation of risk estimates from observed data to
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1 exposure levels of interest (i.e., margin of exposure) and its implications for certainty
2 or uncertainty in quantifying risk;
3 • significant strengths and limitations of the data and analyses, including any major peer
4 reviewers' issues;
5 • appropriate comparison with similar EPA risk analyses or common risks with which
6 people may be familiar; and
7 • comparison with assessment of the same problem by another organization.
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22 U.S. Environmental Protection Agency. (1991d) Guidelines for Developmental Toxicity Risk Assessment. Federal
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24 U.S. Environmental Protection Agency. (1992a) Guidelines for Exposure Assessment. Federal Register
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26 U.S. Environmental Protection Agency. (1992b) Draft report: A Cross-Species Scaling Factor for Carcinogen Risk
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6 U.S. Environmental Protection Agency. (1994b) Report on the Workshop on Cancer Risk Assessment Guidelines
7 Issues. Office of Research and Development, Risk Assessment Forum, Washington, DC. EPA/630/R-94/005a.
8 U.S. Environmental Protection Agency. (1994c) Methods for Derivation of Inhalation Reference Concentrations
9 and Application of Inhalation Dosimetry. Office of Health and Environmental Assessment, Environmental Criteria
10 and Assessment Office, Research Triangle Park, NC. EPA/600/8-90/066F.
11 U.S. Environmental Protection Agency. (1995) Policy for Risk Characterization. Memorandum of Carol M.
12 Browner, Administrator, March 21, 1995, Washington, DC.
13 U.S. Environmental Protection Agency. (1996a) Comparison of the effects of chemicals with combined perinatal
14 and adult exposure vs. adult only exposure in carcinogenesis bioassays.
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19 U.S. Environmental Protection Agency. (1997a) A proposed OPP Policy on Determining the Need for Perinatal
20 Carcinogenicity Testing on a Pesticide. Office of Pesticide Programs, 14 August 1997.
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22 Connection With the Criteria for Requiring In-utero Cancer Studies. Office of Pesticide Programs. FIFRA
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25 U.S. Environmental Protection Agency. (1997c) Exposure factors handbook. EPA/600/P-95/002F.
26 U.S. Environmental Protection Agency. (1998a) Assessment of Thyroid Follicular Cell Tumors. Office of
27 Research and Development, Risk Assessment Forum, Washington, DC. EPA/630/R-97/002.
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29 Office of Science Policy, Washington, DC. EPA 100-B-98-001
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8 immunological, and hematological effects in workers in an ethylene oxide manufacturing plant. Br. J. Indust. Med.
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19 Wellstein, A., Lupu, R., Zugmaier, G., Flamm, S.L., Cheville, A.L., Bovi, P.D., Basicico, C., Lippman, M.E.,
20 Kern, F.G. (1990) Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous
21 basic fibroblast growth factor. Cell Growth Differ. 1:63-71.
22 West, G.B., Brown, J.H., Enquist, B.J. (1997) A General Model for the Origin of Allometric Scaling Laws in
23 Biology. Science 276:122-126. Technical Comments and Responses: www.sciencemag.org/cgi/content/
24 full/s281/5378/75 la
25 Woo, Y.T., Arcos, J.C. (1989) Role of structure-activity relationship analysis in evaluation of pesticides for
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27 and relationship. ACS Symposium Series No. 414. San Diego: Academic Press, pp. 175-200.
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APPENDIX A. WEIGHT-OF-EVIDENCE NARRATIVES
This appendix contains several general illustrations of weight-of-evidence narratives.
NARRATIVE #1
Substance #1
CAS# XXX
CANCER HAZARD SUMMARY
Substance 1 is likely to be carcinogenic to humans by all routes of exposure. The weight
of evidence of human carcinogenicity of Substance 1 is based on (a) findings of carcinogenicity in
rats and mice of both sexes by oral and inhalation exposures; (b) its similarity in structure to other
chlorinated organics that are known to cause liver and kidney damage, and liver and kidney
tumors in rats and mice; (c) suggestive evidence of a possible association between Substance 1
exposure of workers in the laundry and dry cleaning industries and increased cancer risk in a
number of organ systems; and (d) human and animal data indicating that Substance 1 is absorbed
by all routes of exposure.
In comparison with other agents designated as likely carcinogens, the overall weight of
evidence for Substance 1 places it at the low end of the grouping. This is because one cannot
attribute observed excess cancer risk in exposed workers solely to Substance 1. Moreover, there
is considerable scientific uncertainty about the human significance and relevance of certain rodent
tumors associated with exposure to Substance 1 and other chlorinated organics, but insufficient
evidence about mode of action. Hence, the human relevance of the animal evidence of
carcinogenicity relies on a default assumption.
There is no clear evidence about the mode of action for each tumor type induced in rats
and mice. Available evidence suggests that Substance 1 induces cancer mainly by promoting cell
growth rather than via direct mutagenic action, although a mutagenic mode of action for rat
kidney tumors cannot be ruled out. The dos-response assessment should, therefore, adopt both
default approaches, nonlinear and linear. It is recognized that the latter approach likely
overestimates risk at low doses if the mode of action is primarily growth promoting. This
approach, however, may be useful for screening analyses.
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SUPPORTING INFORMATION
Human Data
A number of epidemiologic studies of dry cleaning and laundry workers have reported
elevated incidences of lung, cervix, esophagus, kidney, blood, and lymphoid cancers. Many of
these studies are confounded by coexposure to other petroleum solvents, making them limited for
determining whether the observed increased cancer risks are causally related to Substance 1. The
only investigation of dry cleaning workers with no known exposure to other chemicals did not
evaluate other confounding factors such as smoking, alcohol consumption, and low
socioeconomic status to exclude the possible contribution of these factors to cancer risks.
Animal Data
The carcinogenic potential of Substance 1 has been adequately investigated in two chronic
studies in two rodent species, the first study by gavage and the second study by inhalation.
Substance 1 is carcinogenic in the liver in both sexes of mice when tested by either route of
exposure. It causes marginally increased incidences of mononuclear cell leukemia (MCL) in both
sexes of rats and low incidences of a rare kidney tumor in male rats by inhalation. No increases in
tumor incidence were found in rats treated with Substance 1 by gavage. This rat study was
considered limited because of high mortality of the animals.
Although Substance 1 causes increased incidences of tumors at multiple sites in two
rodent species, controversy surrounds each of the tumor endpoints concerning their relevance
and/or significance to humans (see discussion under Mode of Action).
Other Key Data
Substance 1 is a member of a class of chlorinated organics that often cause liver and
kidney toxicity and carcinogenesis in rodents. Like many chlorinated hydrocarbons, Substance 1
itself has tested negative in a battery of standard genotoxicity tests using bacterial and mammalian
cell systems, including human lymphocytes and fibroblast cells. There is evidence, however, that a
minor metabolite generated by an enzyme found in rat kidney tissue is mutagenic. This kidney
metabolite has been hypothesized to be related to the development of kidney tumors in the male
rat. This metabolic pathway appears to be operative in the human kidney.
Human data indicate that Substance 1 is readily absorbed via inhalation, but to a much
lesser extent by skin contact. Animal data show that Substance 1 is absorbed well by the oral
route.
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MODE OF ACTION
The mechanisms of Substance 1-induced mouse liver tumors are not completely
understood. One mechanism has been hypothesized to be mediated by a genotoxic epoxide
metabolite generated by enzymes found in the mouse liver, but there is a lack of direct evidence in
support of this mechanism. A more plausible mechanism that still needs to be further defined is
related to liver peroxisomal proliferation and toxicity by TCA (trichloroacetic acid), a major
metabolite of Substance 1. However, there are no definitive data indicating that TCA induces
peroxisomal proliferation in humans.
The mechanisms by which Substance 1 induces kidney tumors in male rats are even less
well understood. The rat kidney response may be related to either kidney toxicity or the activity
of a mutagenic metabolite of the parent compound.
The human relevance of Substance 1-induced MCL in rats is unclear. The biological
significance of marginally increased incidences of MCL has been questioned by some, since this
tumor occurs spontaneously in the tested rat strain at very high background rates. On the other
hand, it has been considered by others to be a true finding because there was a decreased time to
onset of the disease and the disease was more severe in treated as compared with untreated
control animals. The exact mechanism by which Substance 1 increases incidence of MCL in rats
is not known.
Overall, there is not enough evidence to justify high confidence in a conclusion about any
single mode of action; it would appear that more than a single mode operates in different rodent
tissues. The apparent lack of mutagenicity of Substance 1 itself and its general growth-promoting
effect on high-background tumors, as well as its toxicity toward mouse liver and rat kidney tissue,
support the view that its predominant mode of action is cell growth promoting rather than
mutagenic. A mutagenic contribution to the renal carcinogenicity due to a metabolite cannot be
entirely ruled out.
NARRATIVE #2
Substance #2
CAS# XXX
CANCER HAZARD SUMMARY
There is suggestive evidence for carcinogenicity of Substance 2, but it is not sufficient for
assessment of human carcinogenic potential.
The evidence on carcinogenicity consists of (a) data from an oral animal study showing a
response only at the highest dose in female rats, with no response in males, and (b) the fact that
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other low-molecular-weight chemicals in this class have shown tumorigenicity in the respiratory
tract after inhalation. The one study of Substance 2 effects by the inhalation route was not
adequately performed. The available evidence is too limited to describe human carcinogenicity
potential or support dose-response assessment.
SUPPORTING INFORMATION
Human Data
An elevated incidence of cancer was reported in a cohort of workers in a chemical plant
who were exposed to a mixture of chemicals, including Substance 2 as a minor component. The
study is considered inadequate because of the small size of the cohort studied and the lack of
adequate exposure data.
Animal Data
In a long-term drinking water study in rats, an increased incidence of adrenal cortical
adenomas was found in the highest dosed females. No other significant finding was made. The
oral rat study was well conducted by a standard protocol. In a 1-year study in hamsters at one
inhalation dose, no tumors were seen. This study was inadequate because of high mortality and
consequent short duration. The chemical is very irritating and is a respiratory toxicant in
mammals. The animal data are too limited for conclusions to be drawn.
Structural Analogue Data
Substance 2's structural analogues, formaldehyde and acetaldehyde, both have
carcinogenic effects on the rat respiratory tract.
Other Key Data
The weight of results of mutagenicity tests in bacteria, fungi, fruit flies, and mice leads to
an overall conclusion of not mutagenic; Substance 2 is lethal to bacteria to a degree that makes
testing difficult and test results difficult to interpret. The chemical is readily absorbed by all
routes.
MODE OF ACTION
Data are not sufficient to judge whether there is a carcinogenic mode of action.
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NARRATIVE #3
Substance #3
CAS# XXX
CANCER HAZARD SUMMARY
Substance 3 is carcinogenic to humans by all routes of exposure. Although several
studies in workers fall short of establishing causality, when considered together, suggest an
elevated risk of lung cancer after long-term exposure to Substance 3. More importantly, animal
cancer bioassay studies and mechanistic studies in both animals and exposed humans have
provided strong consistent results that support a level of concern equal to having conclusive
epidemiologic evidence. The weight of evidence of human carcinogenicity of Substance 3 is based
on (a) consistent evidence of carcinogenicity at multiple sites in both sexes of rats and mice by
oral and inhalation exposure; (b) epidemiologic evidence suggestive of a possible association
between exposure of industrial workers to Substance 3 and elevated risk of lung cancer, which is
the tumor type consistently found in different test species and with different routes of
administration; (c) mutagenic effects in numerous in vivo and in vitro test systems, which are
similar to those found in humans; (d) a similar profile of p53 mutations in transgenic rodent and
human lung tumor tissue; (e) membership in a class of DNA-reactive compounds that are
regularly observed to cause carcinogenic and mutagenic effects in animals. Due to its ready
absorption by all routes of exposure and rapid distribution throughout the body, Substance 3 is
expected to pose a risk by all routes of exposure. The strong evidence of a mutagenic mode of
action supports dose-response assessment that assumes linearity of the relationship.
SUPPORTING INFORMATION
Human Data
Elevated risks of lung cancer different than that associated with smoking have been
reported in exposed workers in several studies. The interpretation of the studies separately is
complicated by the lack of consistency in dose-response, latency period, and average age of
appearance exposures, as well as by exposure to other agents. So, there is no single study that
demonstrates that Substance 3 caused the effects. Nevertheless, several of the studies together
are considered suggestive of Substance 3 carcinogenicity because they consistently show cancer
elevation in the same tissue. Biomonitoring studies of exposed workers find DNA damage in
blood lymphocytes and the degree of DNA damage correlates with the level and duration of
Substance 3 exposure. More importantly, a mechanistic linkage is found for humans by
observation of a similar profile of mutation in the p53 gene from the lung tumor tissue of the p53
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transgenic mouse and exposed workers. This mutation spectra is consistent with the type of
predominant DNA adducts induced by Substance 3. This evidence provides strong support to the
positive suggestion from the worker cancer studies.
Animal Data
Substance 3 causes cancer in multiple tissue sites in rats and mice of both sexes by oral
and inhalation exposure. In particular, there is a consistent trend of a similar tumor site found in
the human studies, namely, an elevated incidence of lung tumor in different species/sexes and by
different routes of exposure. The database is more extensive than usual and the studies are of
good quality. The observation of multisite, multispecies carcinogenic activity by an agent is
considered to be very strong evidence and is often the case with highly mutagenic agents. There
are also strong evidence in many studies showing that Substance 3 is mutagenic across different
phylogenetic levels including rodents, as well as in peripheral cells of exposed humans—a property
that is very highly correlated with carcinogenicity. Further strengthening the concern for human
cancer risk is a similar p53 mutation spectra observed in lung tumor tissue from the p53
transgenic mouse and human cancer biopsies. In humans, a large number of the cases had a
mutation in p53 with a predominance of GC to AT transitions. The mutation spectra of
Substance 3 associated lung tumors differed from patterns reported for sporadic and smoking
related tumors.
Structural Analogue Data
SAR analysis indicates that Substance 3 is a highly DNA-reactive agent. Structurally
related chemicals, also exhibit mutagenic and carcinogenic effects in laboratory animals.
Other Key Data
The structure and DNA reactivity of Substance 3 support potential carcinogenicity. Both
properties are highly correlated with carcinogenicity. Numerous positive mutagenicity tests in
vitro and in vivo add to this support and are reinforced by observation of similar genetic damage
in exposed workers.
Substance 3 is experimentally observed to be readily absorbed by all routes and rapidly
distributed through the body.
MODE OF ACTION
All of the available data in both humans and animals, strongly indicate a mutagenic mode
of action, with a particular human target in lung tissue. A mechanistic linkage is found for rodents
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and humans by observations of a similar profile of mutations in the p53 gene from the lung tumor
tissue of the p53 transgenic mouse and exposed workers. This mutation spectra is consistent with
the type of predominant DNA adducts induced by Substance 3. The tumor suppressor gene, p53,
is a frequently mutated gene in human tumors, including lung. The consistent finding of
mutagencity in experimental assays and human biomonitoring studies, the finding of p53
mutations in transgenic animal and human lung tumor tissue, all points to a mutagenic mode of
action and supports assuming linearity of the dose-response relationship.
NARRATIVE #4
Substance #4
CAS# XXX
CANCER HAZARD SUMMARY
This chemical is likely to be carcinogenic to humans by all routes of exposure. Its
carcinogenic potential is indicated by (a) tumor and toxicity studies on structural analogues, which
demonstrate the ability of the chemical to produce thyroid follicular cell tumors in rats and
hepatocellular tumors in mice following ingestion, and (b) metabolism and hormonal information
on the chemical and its analogues, which contributes to a working mode of action and associates
findings in animals with those in exposed humans. In comparison with other agents designated as
likely carcinogens, the overall weight of evidence for this chemical places it at the lower end of
the grouping. This is because there is a lack of tumor response data on this agent itself.
Biological information on the compound is contradictory in terms of how to quantitate
potential cancer risks. The information on disruption on thyroid-pituitary status argues for using
a margin of exposure evaluation. However, the chemical is an aromatic amine, a class of agents
that are DNA-reactive and induce gene mutation and chromosome aberrations, which argues for
low-dose linearity. Additionally, there is a lack of mode-of-action information on the mouse liver
tumors produced by the structural analogues, also pointing toward a low-dose linear default
approach. In recognition of these uncertainties, it is recommended to quantitate tumors using
both nonlinear (to place a lower bound on the risks) and linear (to place an upper bound on the
risks) default approaches. Given the absence of tumor response data on the chemical per se, it is
recommended that tumor data on close analogues be used to possibly develop toxicity equivalent
factors or relative potencies.
Overall, this chemical is an inferential case for potential human carcinogenicity. The
uncertainties associated with this assessment include (1) the lack of carcinogenicity studies on the
chemical, (2) the use of tumor data on structural analogues, (3) the lack of definitive information
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on the relevance of thyroid-pituitary imbalance for human carcinogenicity, and (4) the different
potential mechanisms that may influence tumor development and potential risks.
SUPPORTING INFORMATION
Human Data
Worker exposure has not been well characterized or quantified, but recent medical
monitoring of workers exposed over a period of several years has uncovered alterations in
thyroid-pituitary hormones (a decrease in T3 and T4 and an increase in TSH) and symptoms of
hypothyroidism. A urinary metabolite of the chemical has been monitored in workers, with
changes in thyroid and pituitary hormones noted, and the changes were similar to those seen in an
animal study.
Animal Data
The concentration of the urinary metabolite in rats receiving the chemical for 28 days was
within twofold of that in exposed workers, a finding associated with comparable changes in
thyroid hormones and TSH levels. In addition, the dose of the chemical given to rats in this study
was essentially the same as that of an analogue that had produced thyroid and pituitary tumors in
rats. The human thyroid responds in the same way as the rodent thyroid following short-term,
limited exposure. Although it is not well established that thyroid-pituitary imbalance leads to
cancer in humans as it does in rodents, information in animals and in exposed humans suggests
similar mechanisms of disrupting thyroid-pituitary function and the potential role of altered TSH
levels in leading to thyroid carcinogenesis.
Structural Analogue Data
This chemical is an aromatic amine, a member of a class of chemicals that has regularly
produced carcinogenic effects in rodents and gene and structural chromosome aberrations in
short-term tests. Some aromatic amines have produced cancer in humans.
Close structural analogues produce thyroid follicular cell tumors in rats and hepatocellular
tumors in mice following ingestion. The thyroid tumors are associated with known perturbations
in thyroid-pituitary functioning. These compounds inhibit the use of iodide by the thyroid gland,
apparently due to inhibition of the enzyme that synthesizes the thyroid hormones (T3, T4).
Accordingly, blood levels of thyroid hormones decrease, which induces the pituitary gland to
produce more TSH, a hormone that stimulates the thyroid to produce more of its hormones. The
thyroid gland becomes larger because of increases in the size of individual cells and their
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proliferation, and upon chronic administration of the chemical, tumors develop. Thus, thyroid
tumor development is significantly influenced by disruption in the thyroid-pituitary axis.
Other Key Data
The chemical can be absorbed by the oral, inhalation, and dermal routes of exposure.
MODE OF ACTION
Data on the chemical and on structural analogues indicate the potential association of
carcinogenesis with perturbation of thyroid-pituitary homeostasis. Structural analogues are
genotoxic, thus raising the possibility of different mechanisms by which this chemical may
influence tumor development.
NARRATIVE #5
Substance #5
CAS# XXX
CANCER HAZARD SUMMARY
Substance 5 is likely to be a human carcinogen by all routes of exposure. Findings are
based on very extensive and significant experimental findings that include (a) tumors at multiple
sites in both sexes of two rodent species via three routes of administration relevant to human
exposure; (b) close structural analogues that produce a spectrum of tumors like those from
Substance 5; (c) significant evidence for the production of reactive Substance 5 metabolites that
readily bind to DNA and produce gene mutations in many systems, including cultured mammalian
and human cells; and (d) two null studies and one positive epidemiologic study; in the positive
study, there may have been exposure to Substance 5. These findings support a decision that
Substance 5 might produce cancer in exposed humans. In comparison to other agents considered
likely human carcinogens, the overall weight of evidence for Substance 5 puts it near the top of
the grouping. Given the agent's mutagenicity, which can influence the carcinogenic process, a
linear dose-response extrapolation is recommended.
Uncertainties include the lack of adequate information on the mutagenicity of Substance 5
in mammals or humans in vivo, although such effects would be expected.
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SUPPORTING INFORMATION
Human Data
The information on the carcinogenicity of Substance 5 from human studies is inadequate.
Two studies of production workers have not shown significant increases in cancer from exposure
to Substance 5 and other chemicals. An increase in lymphatic cancer was reported in a mortality
study of grain elevator workers who may have been exposed to Substance 5 (and other
chemicals).
Animal Data
Substance 5 produced tumors in four chronic rodents studies. Tumor increases were
noted in males and females of rats and mice following oral dermal and inhalation exposure (rat-
oral and two inhalation, mouse—oral and dermal). It produces tumors both at the site of
application (e.g., skin with dermal exposure) and at sites distal to the portal of entry into the body
(e.g., mammary gland) following exposure from each route. Tumors at the same site were noted
in both sexes of a species (blood vessel), both species (forestomach) and via different routes of
administration (lung). Some tumors developed after very short latency, metastasized extensively,
and produced death, an uncommon findings in rodents. The rodent studies were well designed
and conducted except for the oral studies, in which the doses employed caused excessive toxicity
and mortality. However, given the other rodent findings, lower doses would also be anticipated
to be carcinogenic.
Structural Analogue Data
Several chemicals structurally related to Substance 5 are also carcinogenic in rodents.
Among four that are closest in structure, tumors like those seen for Substance 5 were often noted
(e.g., forestomach, mammary, lung), which helps to confirm the findings for Substance 5 itself. In
sum, all of the tumor findings help to establish animal carcinogenicity and support potential human
carcinogenicity for Substance 5.
Other Key Data
Substance 5 itself is not reactive, but from its structure it was expected to be metabolized
to reactive forms. Extensive metabolism studies have confirmed this presumption and have
demonstrated metabolites that bind to DNA and cause breaks in the DNA chain. These lesions
are readily converted to gene mutations in bacteria, fungi, higher plants, insects, and mammalian
and human cells in culture. There are only a limited number of reports on the induction of
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1 chromosome aberrations in mammals and humans; thus far they are negative.
2
3 MODE OF ACTION
4 Human carcinogens often produce cancer in multiple sites of multiple animal species and
5 both sexes and are mutagenic in multiple test systems. Substance 5 satisfies these findings. It
6 produces cancer in males and females of rats and mice. It produces gene mutations in cells across
7 all life forms—plants, bacteria, and animals—including mammals and humans. Given the
8 mutagenicity of Substance 5 exposure and the multiplicity and short latency of Substance 5 tumor
9 induction, it is reasonable to use a linear approach for cancer dose-response extrapolation.
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APPENDIX B. RESPONSES TO THE NATIONAL ACADEMY OF SCIENCES
NATIONAL RESEARCH COUNCIL REPORT SCIENCE AND JUDGMENT IN RISK
ASSESSMENT (N RC, 1994)
Recommendations of the National Academy of Sciences National Research Council
In 1994, the National Academy of Sciences published a report, Science and Judgment in
Risk Assessment. The report was written by a Committee on Risk Assessment of Hazardous Air
Pollutants formed under the Academy's Board on Environmental Studies and Toxicology,
Commission on Life Sciences, National Research Council. The report was called for under
Section 112(o)(l)(A,B) of the Clean Air Act Amendments of 1990, which provided for the EPA
to arrange for the Academy to review:
• risk assessment methodology used by EPA to determine the carcinogenic risk
associated with exposure to hazardous air pollutants from source categories and
subcategories subject to the requirements of this section, and
• improvements in such methodology.
Under Section 112(o)(2)(A,B), the Academy was to consider the following in its review:
• the techniques used for estimating and describing the carcinogenic potency to humans
of hazardous air pollutants, and
• the techniques used for estimating exposure to hazardous air pollutants (for
hypothetical and actual maximally exposed individuals as well as other exposed
individuals).
To the extent practicable, the Academy was also to review methods of assessing adverse human
health effects other than cancer for which safe thresholds of exposure may not exist [Section
112(o)(3)]. The Congress further provided that the EPA Administrator should consider, but need
not adopt, the recommendations in the report and the views of the EPA Science Advisory Board
with respect to the report. Prior to the promulgation of any standards under Section 112(f), the
Administrator is to publish revised guidelines for carcinogenic risk assessment or a detailed
explanation of the reasons that any recommendations contained in the report will not be
implemented [Section 112(o)(6)].
The following discussion addresses the recommendations of the 1994 report that are
pertinent to the EPA cancer risk assessment guidelines. Guidelines for assessment of exposure, of
mixtures, and of other health effects are separate EPA publications. Many of the
recommendations were related to practices specific to the exposure assessment of hazardous air
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pollutants, which are not covered in cancer assessment guidelines. Recommendations about these
other guidelines or practices are not addressed here.
Hazard Classification
The 1994 report contains the following recommendation about classifying cancer hazard:
• The EPA should develop a two-part scheme for classifying evidence on
carcinogenicity that would incorporate both a simple classification and a narrative
evaluation. At a minimum, both parts should include the strength (quality) of the
evidence, the relevance of the animal model and results to humans, and the relevance
of the experimental exposures (route, dose, timing, and duration) to those likely to be
encountered by humans.
The report also presented a possible matrix of 24 boxes that would array weights of evidence
against low, medium, or high relevance, resulting in 24 codes for expressing the weight and
relevance.
These guidelines adopt five standard hazard descriptors and a narrative for presentation of
the weight-of-evidence findings. The descriptors are used within the narrative. There is no
matrix of alphanumerical weight-of-evidence boxes.
The issue of an animal model that is not relevant to humans has been dealt with by not
including an irrelevant response in the weighing of evidence, rather than by creating a weight of
evidence and then appending a discounting factor as the NRC scheme would do. The issue of
relevance is more complex than the NRC matrix makes apparent. Often the question of relevance
of the animal model applies to a single tumor response, but one encounters situations in which
there are more tumor responses in animals than the questioned one. Dealing with this complexity
is more straightforward if it is done during the weighing of evidence rather than after as in the
NRC scheme. Moreover, the same experimental data are involved in deciding on the weight of
evidence and the relevance of a response. It would be awkward to go over the same data twice.
In recommending that the relevance of circumstances of human exposure be taken into
account, the NRC appears to assume that all of the actual conditions of human exposure will be
known when the classification is done. This is not the case. More often than not, the hazard
assessment is applied to risks associated with exposure to different media or environments at
different times. In some cases, there is no priority to obtaining exposure data until the hazard
assessment has been done. The approach of these guidelines is to characterize hazards as to
whether their expression is intrinsically limited by route of exposure or by reaching a particular
dose range based strictly on toxicological and other biological features of the agent. Both the use
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of descriptors and the narrative specifically capture this information. Other aspects of appropriate
application of the hazard and dose-response assessment to particular human exposure scenarios
are dealt with in the characterization of the dose-response assessment, e.g., the applicability of the
dose-response assessment to scenarios with differing frequencies and durations.
The NRC scheme apparently intended that the evidence would be weighed, then given a
low, medium, or high code for some combination of relevance of the animal response, route of
exposure, timing, duration, or frequency. The 24 codes contain none of this specific information,
and, in fact, do not communicate what the conclusion is about. To make the codes communicate
the information apparently intended would require some multiple of the 24 in the NRC scheme.
As the number of codes increases, their utility for communication decreases.
Another reason for declining to use codes is that they tend to become outdated as research
reveals new information that was not contemplated when they were adopted. This has been the
case with the classification system under the 1986 EPA guidelines.
Even though these guidelines do not adopt a matrix of codes, their method of using
descriptors and narratives captures the information the NRC recommended as the most important,
and in the EPA's view, in a more transparent manner.
Dose-Response
The 1994 report contains the following recommendations about dose-response issues:
• EPA should continue to explore, and when scientifically appropriate, incorporate
toxicokinetic models of the link between exposure and biologically effective dose (i.e.,
dose reaching the target tissue).
• Despite the advantages of developing consistent risk assessments between agencies by
using common assumptions (e.g., replacing surface area with body weight to the 0.75
power), EPA should indicate other methods, if any, that would be more accurate.
• EPA should continue to use the linearized multistage model as a default option but
should develop criteria for determining when information is sufficient to use an
alternative extrapolation model.
• EPA should continue to use as one of its risk characterization metrics upper-bound
potency estimates of the probability of developing cancer due to lifetime exposure.
Whenever possible, this metric should be supplemented with other descriptions of
cancer potency that might more adequately reflect the uncertainty associated with the
estimates.
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• EPA should adopt a default assumption for differences in susceptibility among humans
in estimating individual risks.
• In the analysis of animal bioassay data on the occurrence of multiple tumor types, the
cancer potencies should be estimated for each relevant tumor type that is related to
exposure, and the individual potencies should be summed for those tumors.
Toxicokinetic models are encouraged in these guidelines, with discussion of appropriate
considerations for their use. When there are questions as to whether such a model is more
accurate in a particular case than the default method for estimating the human equivalent dose,
both alternatives may be used. It should be noted that the default method for inhalation exposure
is a toxicokinetic model.
The rationale for adopting the oral scaling factor of body weight to the 0.75 power has
been discussed above in the explanation of major defaults. The empirical basis is further explored
in U.S. EPA (1992b). The more accurate approach is to use a toxicokinetic model when data
become available, or to modify the default when data are available, as encouraged under these
guidelines. As the U.S. EPA (1992b) discussion explores in depth, data on the differences among
animals in response to toxic agents are basically consistent with using a power of 1.0, 0.75, or
0.66. The Federal agencies chose the power of 0.75 for the scientific reasons given in the
previous discussion of major defaults; these were not addressed specifically in the NRC report. It
was also considered appropriate, as a matter of policy, for the agencies to agree on one factor.
Again, the default for inhalation exposure is a model that is constructed to become better as more
agent-specific data become available.
EPA proposes not to use a computer model such as the linearized multistage model as a
default for extrapolation below the observed range. The reason is that the basis for default
extrapolation is a theoretical projection of the likely shape of the curve, considering mode of
action. For this purpose, a computer model looks more sophisticated than a straight-line
extrapolation, but is not. The extrapolation will be by straight line as explained in the explanation
of major defaults. This was also recommended by workshop reviewers of a previous draft of
these guidelines (U.S. EPA, 1994b). In addition, a margin-of-exposure analysis is proposed in
cases in which the curve is thought to be nonlinear, based on mode of action. In both cases, the
observed range of data will be modeled by curve fitting in the absence of supporting data for a
biologically based or case-specific model.
The result of using straight-line extrapolation is thought to be an upper bound on low-
dose potency to the human population in most cases, but as discussed in the major defaults
section, it may not always be. Exploration and discussion of uncertainty of parameters in curve-
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fitting a model of the observed data or in using a biologically based or case-specific model is
called for in the dose-response assessment and characterization sections of these guidelines.
The issue of a default assumption for human differences in susceptibility has been
addressed under the major defaults discussion in Section 1.3 with respect to margin-of-exposure
analysis. EPA has considered but decided not to adopt a quantitative default factor for human
differences in susceptibility when a linear extrapolation is used. In general, EPA believes that
linear extrapolation is sufficiently conservative to protect public health. Linear approaches (both
LMS and straight-line extrapolation) from animal data are consistent with linear extrapolation on
the same agents from human data (Goodman and Wilson, 1991; Hoel and Portier, 1994). If
actual data on human variability in sensitivity are available they will, of course, be used.
In analyzing animal bioassay data on the occurrence of multiple tumor types, these
guidelines outline a number of biological and other factors to consider. The objective is to use
these factors to select response data (including nontumor data as appropriate) that best represent
the biology observed. As stated in Section 3 of the guidelines, appropriate options include use of
a single data set, combining data from different experiments, showing a range of results from
more than one data set, showing results from analysis of more than one tumor response based on
differing modes of action, representing total response in a single experiment by combining animals
with tumors, or a combination of these options. The approach judged to best represent the data is
presented with the rationale for the judgment, including the biological and statistical
considerations involved. EPA has considered the approach of summing tumor incidences and
decided not to adopt it. While multiple tumors may be independent, in the sense of not arising
from metastases of a single malignancy, it is not clear that they can be assumed to represent
different effects of the agent on cancer processes. In this connection, it is not clear that summing
incidences provides a better representation of the underlying mode(s) of action of the agent than
combining animals with tumors or using another of the several options noted above. Summing
incidences would result in a higher risk estimate, a step that appears unnecessary without more
reason.
Risk Characterization
• When EPA reports estimates of risk to decisionmakers and the public, it should
present not only point estimates of risk, but also the sources and magnitudes of
uncertainty associated with these estimates.
• Risk managers should be given characterizations of risk that are both qualitative and
quantitative, i.e., both descriptive and mathematical.
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• EPA should consider in its risk assessments the limits of scientific knowledge, the
remaining uncertainties, and the desire to identify errors of either overestimation or
underestimation.
In part as a response to these recommendations, the Administrator of EPA issued
guidelines for risk characterization and required implementation plans from all programs in EPA
(U.S. EPA, 1995). The Administrator's guidance is followed in these cancer guidelines. The
assessments of hazard, dose-response, and exposure will all have accompanying technical
characterizations covering issues of strengths and limitations of data and current scientific
understanding, identification of defaults utilized in the face of gaps in the former, discussions of
controversial issues, and discussions of uncertainties in both their qualitative and, as practicable,
their quantitative aspects.
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APPENDIX C CASE STUDY EXAMPLES FOR HAZARD EVALUATION
This section provides examples of substances that fit the descriptors above. These
examples are based on available information about real substances and are selected to illustrate the
principles for weight-of-evidence evaluation and the application of the classification scheme.
These case studies show the interplay of differing lines of evidence in making a conclusion.
Some particularly illustrate the role that "other key data" can play in conclusions.
Example 1: "Carcinogenic to Humans"—Route-Dependent/Linear Extrapolation
Human Data
Substance 1 is an aluminosilicate mineral that exists in nature with a fibrous habit. Several
descriptive epidemiologic studies have demonstrated very high mortality from malignant
mesothelioma, mainly of the pleura, in three Turkish villages where there was a contamination of
this mineral and where exposure had occurred from birth. Both sexes were equally affected and
at an unusually young age.
Animal Data
Substance 1 has been studied in a single long-term inhalation study in rats at one exposure
concentration that showed an extremely high incidence of pleural mesothelioma (98% in treated
animals versus 0% in concurrent controls). This is a rare malignant tumor in the rat and the onset
of tumors occurred at a very early age (as early as 1 year). Several studies involving injection into
the body cavities of rats or mice (i.e., pleural or peritoneal cavities) also produced high incidences
of pleural or peritoneal mesotheliomas. No information is available on the carcinogenic potential
of substance 1 in laboratory animals via oral and dermal exposures.
Other Key Data
Information on the physical and chemical properties of substance 1 indicates that it is
highly respirable to humans and laboratory rodents. It is highly insoluble and is not likely to be
readily degraded in biological fluid.
No information is available on the deposition, translocation, retention, lung clearance, and
excretion of the substance after inhalation exposure or ingestion. Lung burden studies have
shown the presence of elevated levels of the substance in lung tissue samples of human cases of
pleural mesotheliomas from contaminated villages compared with control villages.
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No data are available on genetic or related effects in humans. The substance has been
shown to induce unscheduled DNA synthesis in human cells in vitro and transformation and
unscheduled DNA synthesis in mouse cells.
The mechanisms by which this substance causes cancer in humans and animals are not
understood, but appear to be related to its unique physical, chemical, and surface properties. Its
fiber morphology is similar to a known group of naturally occurring silicate minerals that have
been known to cause respiratory cancers in humans(including pleural mesothelioma) from
inhalation exposure and genetic changes.
Evaluation
Human evidence is judged to establish a causal link between exposure to substance 1 and
human cancer. Even though the human evidence does not satisfy all criteria for causality, this
judgment is based on a number of unusual observations: large magnitude of the association,
specificity of the association, demonstration of environmental exposure, biological plausibility,
and coherence based on the entire body of knowledge of the etiology of mesothelioma.
Animal evidence demonstrates a causal relationship between exposure and cancer in
laboratory animals. Although available data are not optimal in terms of design (e.g., the use of
single dose, one sex only), the judgment is based on the unusual findings from the only inhalation
experiment in rats (i.e., induction of an uncommon tumor, an extremely high incidence of
malignant neoplasms, and onset of tumors at an early age). Additional evidence is provided by
consistent results from several injection studies showing an induction of the same tumors by
different modes of administration in more than one species.
Other key data, while limited, support the human and animal evidence of carcinogenicity.
It can be inferred from human and animal data that this substance is readily deposited in the
respiratory airways and deep lung and is retained for extended periods of time after first exposure.
Information on related fibrous substances indicates that the modes of action are likely mediated by
the physical and chemical characteristics of the substance (e.g., fiber shape, high aspect ratio, a
high degree of insolubility in lung tissues).
Insufficient data are available to evaluate the human carcinogenic potential of substance 1
by oral exposure. Even though there is no information on its carcinogenic potential via dermal
uptake, it is not expected to pose a carcinogenic hazard to humans by that route because it is very
insoluble and is not likely to penetrate the skin.
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Conclusion
It is concluded that substance 1 is carcinogenic to humans by inhalation exposure. The
weight of evidence of human carcinogenicity is based on (a) exceptionally increased incidence of
malignant mesothelioma in epidemiologic studies of environmentally exposed human populations;
(b) significantly increased incidence of malignant mesothelioma in a single inhalation study in rats
and in several injection studies in rats and mice; and (c) supporting information on related fibrous
substances that are known to cause cancer via inhalation and genetic damage in exposed
mammalian and human mesothelial cells. The human carcinogenic potential of substance 1 via
oral exposure cannot be determined on the basis of insufficient data. It is not likely to pose a
carcinogenic hazard to humans via dermal uptake because it is not anticipated to penetrate the
skin.
The mode of action of this substance is not understood. In addition to this uncertainty,
dose-response information is lacking for both human and animal data. Epidemiologic studies
contain observations of significant excess cancer risks at relatively low levels of environmental
exposure. The use of linear extrapolation in a dose-response relationship assessment is
appropriate as a default since mode-of-action data are not available.
Example 2: "Carcinogenic to Humans"— Any Exposure Conditions/Linear Extrapolation
Human Data
Substance 2 is an alkylating agent that is used extensively as a chemical intermediate in
organic synthesis, particularly in the synthesis of plastics and resins. Several cohort studies of
workers using substance 2 have been conducted. Four studies of chemical workers exposed to
substance 2 (as well as other agents) found an increased mortality rate from lung cancer. The
excess was primarily found in small subgroups with high-level exposure. Although smoking was a
confounding factor, the predominant lung tumor found was small-cell carcinoma, which is distinct
from the squamous cell carcinomas usually found in smokers. Although the type of lung cancer
was consistent among the four studies, the dose-response, latency period, and average age of
appearance was not consistent. Furthermore, there are confounding exposures to other
chemicals. No increase in mortality rate was observed in two studies, one of which had exposures
higher than the studies reporting an increased incidence of lung cancer.
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Animal Data
A multisite tumor response in rats and mice of both sexes is found in 2-year rodent
bioassay studies when substance 2 is administered by various routes. In particular, the induction
of lung tumors is consistently found across different studies, species, and routes of administration.
For example, when administered by inhalation, substance 2 induced a dose-related increase in the
incidences of lung tumors in female and male mice (B6C3F1); and squamous cell carcinomas of
the lung and nasal tumors in male rats (F344). When administered by subcutaneous injection,
substance 2 induced a statistically significant response for pulmonary tumors and local
fibrosarcomas in mice of both sexes. An oral gavage 2-year study resulted in an elevated
incidence of lung tumors in male rats and both sexes of mice, forestomach tumors in both sexes of
rats and mice, liver tumors in both sexes of rats, and urinary bladder tumors in both sexes of mice.
Substance 2 produced lung and forestomach tumors in the p53 mouse cancer transgenic assay
when administered via gavage. It is an initiator of skin tumors in mice.
Other Key Data
Substance 2 is a liquid but can exist as a vapor at room temperature given its high vapor
pressure. It is readily absorbed dermally. Studies in rats indicate that, once absorbed, substance
2 is uniformly distributed throughout the body. It is metabolized by hydrolysis and by conjugation
with glutathione. The ability to form glutathione conjugate varies across animal species, with the
rat being most active, followed by mice.
Substance 2 induces cell transformation in the Syrian Hamster Embryo assay. It is a
direct-acting alkylating agent and is consistently mutagenic when tested in a variety of
nonmammalian and mammalian assays, including in vivo rodent tests. It has been shown to form
DNA adducts and to produce predominantly GC to AT transitions. Substance 2 produces similar
genetic lesions in rodents and humans. It was found to cause dose-related increases, HPRT
mutations, and chromosome aberrations in peripheral blood lymphocytes of exposed workers. A
similar p53 mutation spectra has been found in lung tumor tissue from the p53 transgenic mouse
and human cancer biopsies. In humans, a large number of the cases had a mutation in p53, with a
predominance of GC to AT transitions. The mutation spectra of substance-2-associated lung
tumors differed from patterns reported for sporadic and smoking-related tumors.
SAR analysis indicates that substance 2 is a highly DNA-reactive agent. Structurally
related chemicals also exhibit mutagenic and carcinogenic effects in laboratory animals.
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Evaluation
Available epidemiologic studies, taken together, suggest that a causal association between
exposure to substance 2 and elevated risk of cancer is plausible. This judgment is based on small
but consistent excesses of lung tumors that are distinct from smoking-related lung cancer in the
studies of highly exposed workers. The evidence is close and indicates that causal interpretation
is credible, but not conclusively demonstrated because of certain inconsistencies in the available
studies, possible bias, and confounding factors that could not be adequately excluded.
Extensive evidence indicates that substance 2 is carcinogenic to laboratory animals in
multiple species and at multiple tissue sites with multiple routes of exposure. There is an
induction of malignant tumors to an unusual degree with regard to incidence. In particular, there
is a consistent dose-related induction of lung tumors across different species and routes of
administration in well-designed and conducted studies. This tumor response is similar to that
reported in exposed humans.
The potential human carcinogenicity of substance 2 is reinforced by observations of similar
genetic damage (DNA adducts, HPRT mutations, chromosomal aberrations) in experimental tests
and exposed workers. The genetic effects induced in experimental animals are dose related and
observed at exposures lower than those that produce lung tumors in rodent bioassays. A
mechanistic linkage is found for rodents and humans by observations of a similar profile of
mutations in the p53 gene from the lung tumor tissue of the p53 transgenic mouse and exposed
workers. This mutation spectra is consistent with the type of predominant DNA adducts induced
by substance 2.
Substance 2 belongs to a well-defined, structurally related class of substances whose
members are carcinogenic in rodents and are likely to be human carcinogens.
Conclusion
It is concluded that substance 2 is "carcinogenic to humans" by all routes of exposure.
The weight of evidence of human carcinogenicity is based on (a) consistent evidence of
carcinogenicity in rats and mice by oral and inhalation exposure; (b) epidemiologic evidence
suggestive of a causal association between exposure and elevated risk of lung cancer, which is the
tumor type consistently induced in different test species and with different routes of
administration; (c) evidence of genetic damage in blood lymphocytes of exposed workers; (d)
mutagenic effects in numerous in vivo and in vitro test systems, which are similar to those found
in humans; (e) similar profile of p53 mutations in rodent and human lung tumor tissue; (f)
membership in a class of DNA-reactive compounds that have been shown to cause carcinogenic
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and mutagenic effects in animals; and (g) ability to be absorbed by all routes of exposure,
followed by rapid distribution throughout the body.
The evidence is compelling that the mutagenic properties of substance 2 in experimental
animals and humans are an important influence on the carcinogenic process. Thus, substance 2
acts through a mode of action that is operative in humans and would therefore reasonably be
anticipated to cause cancer in humans. A linear extrapolation should be assumed in dose-response
assessment.
Example 3: "Likely Human Carcinogen"—Any Exposure Conditions/Linear Extrapolation
Human Data
Substance 3 is a brominated alkane. Three studies have investigated the cancer mortality
of workers exposed to this substance. No statistically significant increase in cancer at any site was
found in a study of production workers exposed to substance 3 and several other chemicals.
Elevated cancer mortality was reported in a much smaller study of production workers. An
excess of lymphoma was reported in grain workers who may have had exposure to substance 3
and other chemical compounds. These studies are considered inadequate due to their small
cohort size; lack of or poorly characterized exposure concentrations; or concurrent exposure of
the cohort to other potential or known carcinogens.
Animal Data
The potential carcinogenicity of substance 3 has been extensively studied in an oral gavage
study in rats and mice of both sexes, two inhalation studies of rats of different strains of both
sexes, an inhalation study in mice of both sexes, and a skin painting study in female mice.
In the oral study, increased incidences of squamous-cell carcinoma of the forestomach
were found in rats and mice of both sexes. Additionally, there were increased incidences of liver
carcinomas in female rats, hemangiosarcomas in male rats, and alveolar/bronchiolar adenoma of
the lung of male and female mice. Excessive toxicity and mortality were observed in the rat study,
especially in the high-dose groups, which resulted in early termination of the study, and similar
time-weighted average doses for the high- and low-treatment groups.
In the first inhalation study in rats and mice, increased incidences of carcinomas and
adenocarcinomas of the nasal cavity and hemangio sarcoma of the spleen were found in exposed
animals of each species of both sexes. Treated female rats also showed increased incidences of
alveolar/bronchiolar carcinoma of the lung and mammary gland fibroadenomas. Treated male rats
showed an increased incidence of peritoneal mesothelioma. In the second inhalation study in rats
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(single exposure only), significantly increased incidences of hemangiosarcoma of the spleen and
adrenal gland tumors were seen in exposed animals of both sexes. Additionally, increased
incidences of subcutaneous mesenchymal tumors and mammary gland tumors were induced in
exposed male and female rats, respectively.
Lifetime dermal application of substance 3 to female mice resulted in significantly
increased incidences of skin papillomas and lung tumors.
Several chemicals structurally related to substance 3 are also carcinogenic in rodents. The
spectrum of tumor responses induced by related substances was similar to those seen with
substance 3 (e.g., forestomach, mammary gland, and lung tumors).
Other Key Data
Substance 3 exists as a liquid at room temperature and is readily absorbed by ingestion,
inhalation, and dermal contact. It is widely distributed in the body and is eliminated in the urine
mainly as metabolites (e.g., glutathione conjugate).
Substance 3 is not itself DNA-reactive, but is biotransformed to reactive metabolites, as
inferred by findings of its covalent binding to DNA and induction of DNA strand breaks, both in
vivo and in vitro. Substance 3 has been shown to induce sister chromatid exchanges, mutations,
and unscheduled DNA synthesis in human and rodent cells in vitro. Reverse and forward
mutations have been consistently produced in bacterial assays and in vitro assays using eukaryotic
cells. Substance 3, however, did not induce dominant lethal mutations in mice or rats, or
chromosomal aberrations or micronuclei in bone marrow cells of mice treated in vivo.
Evaluation
Available epidemiologic data are considered inadequate for an evaluation of a causal
association of exposure to the substance and excess of cancer mortality due to major study
limitations.
There is extensive evidence that substance 3 is carcinogenic in laboratory animals.
Increased incidences of tumors at multiple sites have been observed in multiple studies in two
species of both sexes with different routes of exposure. It induces tumors both at the site of entry
(e.g., nasal tumors via inhalation, forestomach tumors by ingestion, skin tumors with dermal
exposure) and at distal sites (e.g., mammary gland tumors). Additionally, it induced tumors at the
same sites in both species and sexes via different routes of exposure (e.g., lung tumors). With the
exception of the oral study in which the employed doses caused excessive toxicity and mortality,
the other studies are considered adequately designed and well conducted. Overall, given the
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magnitude and extent of animal carcinogenic responses to substance 3, coupled with similar
responses to structurally related substances, these animal findings are judged to be highly relevant
and predictive of human responses.
Other key data, while not very extensive, are judged to be supportive of carcinogenic
potential. Substance 3 has consistently been shown to be mutagenic in mammalian cells, including
human cells, and in nonmammalian cells; thus, mutation is likely a mode of action for its
carcinogenic activity. However, the possible involvement of other modes of action has not been
fully investigated. Furthermore, induction of genetic changes from in vivo exposure to substance
3 has not been demonstrated.
Conclusion
Substance 3 is likely to be carcinogenic to humans. In comparison with other agents
designated as likely human carcinogens, the overall weight of evidence for substance 3 puts it at
the high end of the grouping.
The weight of evidence of human carcinogenicity is based on animal evidence and other
key evidence. Human data are inadequate for an evaluation of human carcinogenicity. The
overall weight of evidence is based on (a) extensive animal evidence showing induction of
increases of tumors at multiple sites in both sexes of two rodent species via three routes of
administration relevant to human exposure; (b) tumor data of structural analogues exhibiting
similar patterns of tumors in treated rodents; (c) in vitro evidence for mutagenic effects in
mammalian cells and nonmammalian systems; and (d) its ability to be absorbed by all routes of
exposure followed by rapid distribution throughout the body.
Some uncertainties are associated with the mechanisms of carcinogenicity of substance 3.
Although there is considerable evidence indicating that mutagenic events could account for
carcinogenic effects, there is still a lack of adequate information on the mutagenicity of substance
3 in vivo in animals or humans. Moreover, alternative modes of action have not been explored.
Nonetheless, available data indicate a likely mutagenic mode of action. Linear extrapolation
should be assumed in dose-response assessment.
Example 4: "Likely Human Carcinogen "—All Routes/Linear and Nonlinear Extrapolation
Human Data
Substance 4 is a chlorinated alkene solvent. Several cohort studies of dry cleaning and
laundry workers exposed to substance 4 and other solvents reported significant excesses of
mortality due to cancers of the lung, cervix, esophagus, kidney, bladder, lymphatic and
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hematopoietic system, colon, or skin. No significant cancer risks were observed in a subcohort of
one of these investigations of dry cleaning workers exposed mainly to substance 4. Possible
confounding factors such as smoking, alcohol consumption, or low socioeconomic status were
not considered in the analyses of these studies.
A large case-control study of bladder cancer did not show any clear association with dry
cleaning. Several case-control studies of liver cancer identified an increased risk of liver cancer
with occupational exposure to organic solvents. The specific solvents to which workers were
exposed and exposure levels were not identified.
Animal Data
The potential carcinogenicity of substance 4 has been investigated in two long-term
studies in rats and mice of both sexes by oral administration and inhalation.
Significant increases in hepatocellular carcinomas were induced in mice of both sexes
treated with substance 4 by oral gavage. No increases in tumor incidence were observed in
treated rats. Limitations in both experiments included control groups smaller than treated groups,
numerous dose adjustments during the study, and early mortality due to treatment-related
nephropathy.
In the inhalation study, there were significantly increased incidences of hepatocellular
adenoma and carcinoma in exposed mice of both sexes. In rats of both sexes, there were
marginally significant increased incidences of mononuclear cell leukemia (MCL) when compared
with concurrent controls. The incidences of MCL in control animals, however, were higher than
historical controls from the conducting laboratory. The tumor finding was also judged to be
biologically significant because the time to onset of tumor was decreased and the disease was
more severe in treated than in control animals. Low incidences of renal tubular cell adenomas or
adenocarcinomas were also observed in exposed male rats. The tumor incidences were not
statistically significant, but there was a significant trend.
Other Key Data
Substance 4 has been shown to be readily and rapidly absorbed by inhalation and ingestion
in humans and laboratory animals. Absorption by dermal exposure is slow and limited. Once
absorbed, substance 4 is primarily distributed to and accumulated in adipose tissue and the brain,
kidney, and liver. A large percentage of substance 4 is eliminated unchanged in exhaled air, with
urinary excretion of metabolites comprising a much smaller percentage. The absorption and
distribution profiles of substance 4 are similar across species including humans.
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Two major metabolites (trichloroacetic acid [TCA] and trichloroethanol), which are
formed by a P-450-dependent mixed-function oxidase enzyme system, have been identified in all
studied species, including humans. There is suggestive evidence for the formation of an epoxide
intermediate based on the detection of two other metabolites (oxalic acid and trichloroacetyl
amide). In addition to oxidative metabolism, substance 4 also undergoes conjugation with
glutathione. Further metabolism by renal beta-lyases could lead to two minor active metabolites
(trichlorovinyl thiol and dichlorothiokente).
Toxicokinetic studies have shown that the enzymes responsible for the metabolism of
substance 4 can be saturated at high exposures. The glutathione pathway was found to be a
minor pathway at low doses, but more prevalent following saturation of the cytochrome P-450
pathway. Comparative in vitro studies indicate that mice have a greater capacity to metabolize to
TCA than rats and humans. Inhalation studies also indicate saturation of oxidative metabolism of
substance 4, which occurs at higher dose levels in mice than in rats and humans. Based on these
findings, it has been postulated that the species differences in the carcinogenicity of substance 4
between rats and mice may be related to the differences in the metabolism to TCA and glutathione
conjugates.
Substance 4 is a member of the class of chlorinated organics that often cause liver and
kidney toxicity and carcinogenesis in rodents. Like many chlorinated organics, substance 4 itself
does not appear to be mutagenic. Substance 4 was generally negative in in vitro bacterial systems
and in vivo mammalian systems. However, a minor metabolite formed in the kidney by the
glutathione conjugation pathway has been found to be a strong mutagen.
The mechanisms of induced carcinogenic effects of substance 4 in rats and mice are not
completely understood. It has been postulated that mouse liver carcinogenesis is related to liver
peroxisomal proliferation and toxicity of the metabolite TCA. Information on whether or not
TCA induces peroxisomal proliferation in humans is not definitive. The induced renal tumors in
male rats may be related either to kidney toxicity or the activity of a mutagenic metabolite. The
mechanisms of increases in MCL in rats are not known.
Evaluation
Available epidemiologic studies, taken together, provide suggestive evidence of a possible
causal association between exposure to substance 4 and cancer incidence in the laundry and dry
cleaning industries. This is based on consistent findings of elevated cancer risks in several studies
of different populations of dry cleaning and laundry workers. However, each individual study is
compromised by a number of study deficiencies including small numbers of cancers, confounding
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exposure to other solvents, and poor exposure characterization. Others may interpret these
findings collectively as inconclusive.
There is considerable evidence that substance 4 is carcinogenic to laboratory animals. It
induces tumors in mice of both sexes by oral and inhalation exposure and in rats of both sexes via
inhalation. However, owing to incomplete understanding of the mode of action, the predictivity
of animal responses to humans is uncertain.
Animal data of structurally related compounds showing common target organs of toxicity
and carcinogenic effects (but lack of mutagenic effects) provide additional support for the
carcinogenicity of substance 4. Comparative toxicokinetic and metabolism information indicates
that the mouse may be more susceptible to liver carcinogenesis than rats and humans. This may
indicate differences of the degree and extent of carcinogenic responses, but does not detract from
the qualitative weight of evidence of human carcinogenicity. The toxicokinetic information also
indicates that oral and inhalation are the major routes of human exposure.
Conclusion
Substance 4 is likely to be carcinogenic to humans by all routes of exposure. The weight
of evidence of human carcinogenicity is based on: (a) demonstrated evidence of carcinogenicity in
two rodent species of both sexes via two relevant routes of human exposure; (b) the substance's
similarity in structure to other chlorinated organics that are known to cause liver and kidney
toxicity and carcinogenesis in rodents; (c) suggestive evidence of a possible association between
exposure to the substance in the laundry and dry cleaning industries and increased cancer
incidence; and (d) human and animal data indicating that the substance is absorbed by all routes of
exposure.
There is considerable scientific uncertainty about the human significance of certain rodent
tumors associated with substance 4 and related compounds. In this case, the human relevance of
the animal evidence of carcinogenicity relies on the default assumption.
Overall, there is not enough evidence to give high confidence in a conclusion about any
single mode of action; it appears that more than one is plausible in different rodent tissues.
Nevertheless, the lack of mutagenicity of substance 4 and its general growth-promoting effect on
high background tumors, as well as its toxicity toward mouse liver and rat kidney tissue, support
the view that the predominant mode is growth-promoting rather than mutagenic. A mutagenic
contribution to carcinogenicity due to a metabolite cannot be ruled out. The dose-response
assessment should, therefore, adopt both default approaches, nonlinear and linear extrapolations.
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The latter approach is very conservative since it likely overestimates risk at low doses in this case,
and is primarily useful for screening analyses.
Example 5: "Likely/Not Likely Human Carcinogen"—Range of Dose Limited, Margin-of-
Exposure Extrapolation
Human Data
Substance 5 is a metal-conjugated phosphonate. No human tumor or toxicity data exist
on this chemical.
Animal Data
Substance 5 caused a statistically significant increase in the incidence of urinary bladder
tumors in male, but not female, rats at 30,000 ppm (3%) in the diet in a long-term study. Some of
these animals had accompanying urinary tract stones and toxicity. No bladder tumors or adverse
urinary tract effects were seen in two lower dose groups (2,000 and 8,000 ppm) in the same
study. A chronic dietary study in mice at doses comparable to those in the rat study showed no
tumor response or urinary tract effects. A 2-year study in dogs at doses up to 40,000 ppm
showed no adverse urinary tract effects.
Other Key Data
Subchronic dosing of rats confirmed that there was profound development of stones in the
male bladder at doses comparable to those causing cancer in the chronic study, but not at lower
doses. Sloughing of the epithelium of the urinary tract accompanied the stones.
There was a lack of mutagenicity relevant to carcinogenicity. In addition, there is nothing
about the chemical structure of substance 5 to indicate DNA reactivity or carcinogenicity.
Substance 5 is composed of a metal, an ethanol, and a simple phosphorus-oxygen-
containing component. The metal is not absorbed from the gut, whereas the other two
components are absorbed. At high doses, ethanol is metabolized to carbon dioxide, which makes
the urine more acidic; the phosphorus level in the blood and calcium in the urine are increased.
Chronic testing of the phosphorus-oxygen-containing component alone in rats did not show any
tumors or adverse effects on the urinary tract.
Because substance 5 is a metal complex, it is not likely to be readily absorbed from the
skin.
Evaluation
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Substance 5 produced cancer of the bladder and urinary tract toxicity in male, but not
female rats and mice, and dogs failed to show the toxicity noted in male rats. The mode of action
developed from the other key data to account for the toxicity and tumors in the male rats is the
production of bladder stones. At high but not lower subchronic doses in the male rat, substance 5
leads to elevated blood phosphorus levels; the body responds by releasing excess calcium into the
urine. The calcium and phosphorus combine in the urine and precipitate into multiple stones in
the bladder. The stones are very irritating to the bladder; the bladder lining is eroded and cell
proliferation occurs to compensate for the loss of the lining. Cell layers pile up, and finally,
tumors develop. Stone formation does not involve the chemical per se but is secondary to the
effects of its constituents on the blood and, ultimately, the urine. Bladder stones, regardless of
their cause, commonly produce bladder tumors in rodents, especially the male rat.
Conclusion
Substance 5, a metal aliphatic phosphonate, is likely to be carcinogenic to humans only
under high-exposure conditions following oral and inhalation exposure that lead to bladder stone
formation, but is not likely to be carcinogenic under low-exposure conditions. It is not likely to
be a human carcinogen via the dermal route, given that the compound is a metal conjugate that is
readily ionized and its dermal absorption is not anticipated. The weight of evidence is based on
(a) bladder tumors only in male rats; (b) the absence of tumors at any other site in rats or mice; (c)
the formation of calcium-phosphorus-containing bladder stones in male rats at high, but not low,
exposures that erode bladder epithelium and result in profound increases in cell proliferation and
cancer; and (d) the absence of structural alerts or mutagenic activity.
There is a strong mode-of-action basis for the requirements of (a) high doses of substance
5, (b) which lead to excess calcium and increased acidity in the urine, (c) which result in the
precipitation of stones, and (d) the necessity of stones for toxic effects and tumor hazard
potential. Lower doses fail to perturb urinary constituents, lead to stones, produce toxicity, or
give rise to tumors. Therefore, dose-response assessment should assume nonlinearity.
A major uncertainty is whether the profound effects of substance 5 may be unique to the
rat. Even if substance 5 produced stones in humans, there is only limited evidence that humans
with bladder stones develop cancer. Most often human bladder stones are either passed in the
urine or lead to symptoms resulting in their removal. However, since one cannot totally dismiss
the male rat findings, some hazard potential may exist in humans following intense exposures.
Only fundamental research could illuminate this uncertainty.
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Example 6: "Suggestive" Evidence
Human Data
Substance 6 is an unsaturated aldehyde. In a cohort study of workers in a chemical plant
exposed to a mixture of chemicals with substance 6 as a minor component, a greater risk of
cancer was reported than was expected. This study is considered inadequate because of multiple
exposures, small cohort, and poor exposure characterization.
Animal Data
Substance 6 was tested for potential carcinogenicity in a drinking water study in rats, an
inhalation study in hamsters, and a skin painting study in mice. No significant increases in tumors
were observed in male rats treated with substance 6 at three dose levels in drinking water.
However, a significant increase of adrenal cortical adenomas was found in the only treated female
dose group administered a dose equivalent to the high dose of males. This study used a small
number of animals (20 per dose group).
No significant finding was detected in the inhalation study in hamsters. This study is
inadequate due to the use of too few animals, short duration of exposure, and inappropriate dose
selection (use of a single exposure that was excessively toxic as reflected by high mortality).
No increase in tumors was induced in the skin painting study in mice. This study is of
inadequate design for carcinogenicity evaluation because of several deficiencies: small number of
animals, short duration of exposure, lack of reporting about the sex and age of animals, and purity
of test material.
Substance 6 is structurally related to low-molecular-weight aldehydes that generally
exhibit carcinogenic effects in the respiratory tracts of laboratory animals via inhalation exposure.
Three skin painting studies in mice and two subcutaneous injection studies of rats and mice were
conducted to evaluate the carcinogenic potential of a possible metabolite of substance 6
(identified in vitro). Increased incidences of either benign or combined benign and malignant skin
tumors were found in the dermal studies. In the injection studies of rats and mice, increased
incidences of local sarcomas or squamous cell carcinoma were found at the sites of injection. All
of these studies are limited by the small number of test animals, the lack of characterization of test
material, and the use of single doses.
Other Key Data
Substance 6 is a flammable liquid at room temperature. Limited information on its
toxicokinetics indicates that it can be absorbed by all routes of exposure. It is eliminated in the
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urine mainly as glutathione conjugates. Substance 6 is metabolized in vitro by rat liver and lung
microsomal preparations to a dihydroxylated aldehyde.
No data were available on the genetic and related effects of substance 6 in humans. It did
not induce dominant lethal mutations in mice. It induced sister chromatid exchanges in rodent
cells in vitro. The mutagenicity of substance 6 is equivocal in bacteria. It did not induce DNA
damage or mutations in fungi.
Evaluation
Available human data are judged suggestive, but not sufficient for an evaluation of any
causal relationship between exposure to substance 6 and human cancer.
The carcinogenic potential of substance 6 has not been adequately studied in laboratory
animals due to serious deficiencies in study design, especially the inhalation and dermal studies.
There is suggestive evidence of carcinogenicity in the drinking water study in female rats.
However, the significance of that study to a potential for human response is uncertain since the
finding is limited to occurrence of benign tumors in one sex, and at the high dose only. Additional
suggestion for animal carcinogenicity comes from observation that a possible metabolite is
carcinogenic at the site of administration. This metabolite, however, has not been studied in vivo.
Overall, the animal evidence is judged to be suggestive for human carcinogenicity.
Other key data, taken together, do not add significantly to the overall weight of evidence
of carcinogenicity. SAR analysis indicates that substance 6 would be DNA-reactive. However,
mutagenicity data are inconclusive. Limited in vivo data do not support a mutagenic effect.
While there is some evidence of DNA damage in rodent cells in vitro, there is either equivocal or
no evidence of mutagenicity in nonmammalian systems.
Conclusion
While there is a suggestion of animal carcinogenicity, the data are inadequate for a
judgment about the human carcinogenicity potential of substance 6. Both human and animal data
are judged inadequate for an evaluation. There is evidence suggestive of potential carcinogenicity
on the basis of limited animal findings and SAR considerations. Data are not sufficient to judge
whether there is a mode of carcinogenic action. Additional studies are needed for a full evaluation
of the potential carcinogenicity of substance 6. Hence, dose-response assessment is not
appropriate.
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Example 7: "Not Likely to be a Human Carcinogen"—Appropriately Studied Chemical in
Animals Without Tumor Effects
Human Data
Substance 7, a plant extract, has not been studied for its toxic or carcinogenic potential in
humans.
Animal Data
Substance 7 has been studied in four chronic studies in three rodent species. In a feeding
study in rats, males showed a nonsignificant increase in benign tumors of the parathyroid gland in
the high-dose group, where the incidence in concurrent controls greatly exceeded the historical
control range. Females demonstrated a significant increase in various subcutaneous tumors in the
low-dose group, but findings were not confirmed in the high-dose group, and there was no dose-
response relationship. These effects were considered as not adding to the evidence of
carcinogenicity. No tumor increases were noted in a second adequate feeding study in male and
female rats. In a mouse feeding study, no tumor increases were noted in dosed animals. There
was some question as to the adequacy of the dosing; however, it was noted that in the mouse 90-
day subchronic study that a dose of twice the high dose in the chronic study led to significant
decrements in body weight. In a hamster study there were no significant increases in tumors at
any site. No structural analogues of substance 7 have been tested for cancer.
Other Key Data
There are no structural alerts that would suggest that substance 7 is a DNA-reactive
compound. It is negative for gene mutations in bacteria and yeast, but positive in cultured mouse
cells. Tests for structural chromosome aberrations in cultured mammalian cells and in rats are
negative; however, the animals were not tested at sufficiently high doses. Substance 7 binds to
proteins of the cell division spindle; therefore, there is some likelihood for producing numerical
chromosome aberrations, an endpoint that is sometimes noted in cancers. In sum, there is limited
and conflicting information concerning the mutagenic potential of the agent.
The compound is absorbed via oral and inhalation exposure but only poorly via the skin.
Evaluation
The only indication of a carcinogenic effect comes from the finding of benign tumors in
male rats in a single study. There is no confirmation of a carcinogenic potential from dosed
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females in that study, in males and females in a second rat study, or from mouse and hamster
studies.
There is no structural indication that substance 7 is DNA-reactive, there is inconsistent
evidence of gene mutations, and chromosome aberration testing is negative. The agent binds to
cell division spindle proteins and may have the capacity to induce numerical chromosome
anomalies. Further information on gene mutations and in vivo structural and numerical
chromosome aberrations may be warranted.
Conclusion
Substance 7 is not likely to be carcinogenic to humans via all relevant routes of exposure.
This weight-of-evidence judgment is largely based on the absence of significant tumor increases in
chronic rodent studies. Adequate cancer studies in rats, mice, and hamsters fail to show any
carcinogenic effect; a second rat study showed an increase in benign tumors at a site in dosed
males, but not females.
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APPENDIX D. CASE STUDY EXAMPLES FOR MODE-OF-ACTION EVALUATION
This appendix contains case examples to illustrate the application of the framework for
mode-of-action analysis. Evaluations of mode-of-action information will ordinarily appear
before or within the hazard characterization section of a risk assessment. Since these examples
are given outside of a risk assessment, the basic data that underlie the evaluation are
summarizedfirst for reference, followed by the mode-of-action analysis.
D.1.0. EXAMPLE 1: CHEMICAL T (THYROID DISRUPTION)
D.l.l. HAZARD DATA SUMMARY
D.l.1.1. Data Availability
Data include a rat chronic/carcinogenicity feeding study, an 18-month CD-I mouse
carcinogenicity study, a 1-year dog feeding study, a subchronic feeding study in the rat, a 4-week
and 1-year subchronic feeding study in the dog, a 21-day dermal study in the rat, developmental
toxicity studies in the rat and rabbit, a two-generation reproduction study in the rat, mutagenicity
studies, metabolism studies, and special subchronic mechanistic studies.
D.l.l.1.1. Rat
D.l.l. 1.1.1. 24-month toxicity. Male and female Sprague-Dawley rats received chemical T in the
diet for 24 months. Thyroid follicular cell tumor incidence was increased in male but not female
animals (see Table D-l). Tumor incidence in the two high-dose male groups was higher than in
historical control studies. Thyroid and liver weights were increased in the two high-dose groups. A
few renal tubular adenomas occurred in dosed male and female animals, but there was no statistical
significance. SGPT was increased in high-dose animals; some other liver enzymes were increased at
various times.
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Table D-l. Thyroid follicular cell tumor incidence in male rats
Tumor
Dose (ppm in diet)
0
1
10
100
1000
3000a
Benign
1/50 b
2/47
0/49
2/47
8/49
12/48 b
Malignant
1/50 b
1/47
0/49
0/47
1/49
4/48
Combined
2/50 b
3/47
0/49
2/47
9/49
14/48 b
Two animals had both benign and malignant tumors.
bStatistically significant for trend noted at control; pairwise comparison noted at dose level.
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D.l.1.1.1.2. Special subchronic studies. Groups of male Sprague-Dawley rats were fed
chemical T at 3000 ppm in the diet for 7, 14, 28, 56, or 90 days. Starting at 7 days, TSH levels
were significantly increased and T4 values were significantly decreased. There were also
significant increases in thyroid and liver weights and for follicular cell hypertrophy and
hyperplasia. Hepatic UDPGT activity for T4 was increased, while hepatic 5'-monodeiodinase
activity was either unaffected or decreased. Radioiodine uptake into the thyroid gland was
measured. The percent of the dose per gram of thyroid tissue was equivalent in 3000 ppm and
control groups, as was protein-bound iodide per mg of thyroid protein. Activities of hepatic aryl
hydrocarbon hydroxylase, ethoxycoumarin O-dehydrase, and cytochrome P-450 were significantly
increased in chemical T dosed animals.
Groups of male Sprague-Dawley rats were fed chemical T (30, 100, 300, 1000, 3000
ppm) for 56 days; some animals were taken off chemical T for another 56 or 112 days to evaluate
reversibility of effects. Thyroid weights were significantly increased in the top two doses, while
liver weights were increased in the top three doses. T4 UDPGT activity was increased in the top
two doses. T4 was decreased and TSH increased at the top dose, along with increases in the
incidence of follicular cell hypertrophy and hyperplasia. Upon stopping chemical T dosing, all
parameters returned to normal except for thyroid weight. Elimination of radioiodine-labeled T4
from the blood and into the bile was measured after 56 days of chemical T dosing. Blood
clearance was twice as fast in dosed animals as in controls, while there was a 40% increase in the
rate of excretion of the hormone into the bile of treated animals.
D.l.1.1.2. Dog
D.l.1.1.2.1. Subchronic toxicity. Subchronic feeding of chemical T (0, 10, 100, 1000, 5000
ppm) produced an increase in thyroid weight and hyperplasia of the gland at 5000 ppm. There
was hepatocellular hypertrophy at 1000 ppm and above.
D.l. 1.1.2.2. 12-month toxicity. One-year feeding of chemical T (1, 20, 200, 2000 ppm) led to
hepatocellular hypertrophy/hyperplasia at 200 and 2000 ppm but not at 0 or 20 ppm. At 2000
ppm, absolute and relative liver weights were increased. At 2000 ppm, there were increases in
SGOT, SGPT, GGT, and ALK, and decreases in cholesterol, albumin, and total protein.
D.l.1.1.3. Mouse
D.l.1.1.3.1. 18-month toxicity. In an 18-month chemical T feeding study (0, 1, 10, 100, 400,
800 ppm), there were no increases in tumor incidence at any site. Absolute and relative liver
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weights were statistically significantly increased over controls at the highest dose level, as were
kidney weights in the female. Increases in liver enzymes were noted at various intervals, including
SGPT, SGOT, and ALK. Dose levels in the study were considered adequate.
D.l.1.2. Mutagenicity
Negative results were seen in four strains of Salmonella with or without metabolic
activation; negative results in assay of forward mutation of HGPT locus of Chinese hamster ovary
cells (dosing probably not sufficient); negative results in mouse bone marrow micronucleus assay;
negative results in assay for unscheduled DNA synthesis in rat hepatocytes pretreated with
chemical T. The compound does not have a structure that suggests electrophilicity.
D.1.2. SUMMARY DESCRIPTION OF POSTULATED MODE OF ACTION
Thyroid hormone production is regulated by actions of the hypothalamus, pituitary, and
thyroid glands. Homeostasis of thyroid hormone is maintained by a feedback loop among the
hypothalamus and pituitary and the thyroid gland. The hypothalamus produces thyrotrophin
reducing hormone (TRH), which stimulates the pituitary to produce thyroid stimulating hormone
(TSH) which, in turn, stimulates the thyroid to produce thyroid hormone. The hypothalamus and
pituitary respond to a high level of circulating thyroid hormone by suppressing TRH and TSH
production, and to a low level by increasing them. The mode of action considered is continuous
elevation of TSH levels that stimulates the thyroid gland to deplete its stores of thyroid hormone
and continues to push production, resulting in hypertrophy of the production cells (follicular cells)
leading to hyperplasia, nodular hyperplasia and, eventually, tumors of these cells. In rats, the
chain of events may be induced by direct effects on hormone synthesis or by metabolic removal of
circulating hormone.
D.1.3. KEY EVENTS
The key events considered with respect to chemical T-induced tumorigenesis in male rats
include hormone changes in TSH, T4, and T3, and changes in hepatic T4-UDPGT, indicators of
liver microsomal enzyme induction, enhanced liver metabolism, increased biliary excretion of T4,
increase in thyroid weight and liver weight, and thyroid follicular cell hypertrophy/hyperplasia.
These events have been well defined and measured in male rats in subchronic studies, augmenting
observations at interim and terminal sacrifice in a chronic study.
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D.1.4. STRENGTH, CONSISTENCY, SPECIFICITY OF ASSOCIATION OF TUMOR
RESPONSE WITH KEY EVENTS
The thyroid tumor response in the chronic study at the highest dose was associated with
hypertrophy/hyperplasia in the thyroid and increase in weight of the thyroid. In subchronic
studies, the organ weight and hypertrophy/hyperplasia were shown to appear and reverse in
statistically significant degrees under the same conditions of dose and time as the appearance and
reversal of changes in thyroid hormone levels and thyroid hormone metabolism. Stop/recovery
studies showed that cessation of dosing was followed in turn by return of hormone levels to
control levels, reduction in liver and thyroid weights, and reversal of hyperplasia in thyroid
follicular cells. The only sign slow to reverse was thyroid weight after the longest dosing period.
Strength, consistency, and specificity of association were well established in the studies.
D.1.5. DOSE-RESPONSE RELATIONSHIP
Dose correlations exist for parameters in the chronic and subchronic studies for all of the
relevant parameters. Thyroid follicular cell tumors, thyroid hypertrophy/hyperplasia, and
increased thyroid and liver weight are noted at similar doses, usually at dietary levels of 1000 and
3000 ppm chemical T. Correspondingly in the subchronic study, at 3000 ppm T4 is depressed
while TSH is elevated. At 1000 and 3000 ppm, hepatic T4-UDPGT activity is statistically
significantly elevated, and there is an increase in biliary excretion of T4 at 3000 ppm. The only
parameter showing significant effect at a dose below 100 ppm chemical T was liver weight
increase in a subchronic study at 300 ppm.
D.1.6. TEMPORAL ASSOCIATION
The chronic study, together with the three subchronic studies of key events observing
effects after different durations at one dose, at multiple doses, and after recovery, shows events
occurring in the following sequence: (1) increase in hepatic glucuronidation, de-iodination and
excretion of T4, as well as its elimination from the blood; (2) a rise in circulating TSH; (3) an
increase in thyroid weight and thyroid follicular cell hypertrophy; (4) thyroid follicular cell
hyperplasia; and (5) thyroid follicular cell tumors. The stop experiments indicate reversal of the
thyroid and liver weight increases as well as reversal of hormone and other protein measures.
While reversal of thyroid weight increase in the recovery study was less after a longer duration of
treatment, hypertrophy/hyperplasia did reverse after the longer duration.
D.1.7. BIOLOGICAL PLAUSIBILITY AND COHERENCE OF THE DATABASE
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Under EPA science policy (U.S. EPA, 1998a), determination of the antithyroid activity of
a chemical requires empirical demonstration of five items: (1) increases in thyroid growth, (2)
changes in thyroid and pituitary hormones, (3) location of the site(s) of antithyroid action, (4)
dose-response correlations among various key precursor events and tumor incidence, and (5)
reversibility of effects following treatment cessation. The database on chemical T documents all
such information.
Thyroid tumorigenesis, particularly in the male rat, has been observed to be associated
with exposure to a number of industrial chemicals, pesticides, and pharmaceuticals. A significant
number of these appear to work in a manner similar to chemical T, by enhancing thyroid hormone
metabolism and excretion by the liver.
Thyroid tumors did not appear in the female rats in the 2-year study. Thyroid hypertrophy
and hyperplasia were observed in the females 6 months after their appearance in males. As is
noted with other chemicals, the female rat is less sensitive to the effect of antithyroid chemicals
regarding key events and tumor development. Hepatic enlargement and effects are noted in the
mouse and dog studies, as they are in the rat. In addition, dogs receiving high doses of chemical
T show enlargement of the thyroid gland.
D.1.8. OTHER MODES OF ACTION
Chemical T does not belong to a class of chemicals that is expected to generate reactive
metabolites, and no related chemicals have been tested for carcinogenicity. Short-term studies
demonstrate that the chemical does not increase gene mutations in Salmonella (Ames test) or
cultured mammalian cells (maximal dosage may not have been reached), micronuclei in bone
marrow cells, and unscheduled DNA synthesis in cultured cells. No other modes of action, apart
from thyroid disruption, are described to account for the thyroid tumors.
Several sites of action were investigated as being the source of the antithyroid effects of
chemical T. The chemical does not inhibit the entry of inorganic iodide into the thyroid (iodide
pump) or block the organification and incorporation of iodide into thyroid hormone (thyroid
peroxidase); likewise, it does not inhibit monodeiodinase, which blocks the conversion of T4 to
t3.
Chemical T administration leads to renal adenomas in male and female rats; the response
lacked statistical significance. The mode of action for the thyroid tumors does not account for the
renal tumors. Assessment of the significance and mode of action of the renal tumors requires
separate analysis.
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D.1.9. CONCLUSION
The weight of evidence supports a conclusion that chemical T acts by inducing hepatic
metabolism and biliary elimination of thyroid hormone, prompting increased production of TSH,
which ultimately results in thyroid follicular cell neoplasia as postulated.
D.1.10. RELEVANCE OF THE MODE OF ACTION TO HUMANS
Relevance to humans
Chemical T affects the liver of rats, mice, and dogs, and the thyroid of rats and dogs.
Given the breadth of responses, it is possible that humans may respond similarly. The subject of
the relevance of an antithyroid mode of action for thyroid tumors is extensively covered in the
Agency's policy for the assessment of this mode of action (U.S. EPA, 1998a). In summary the
policy states:
The role of thyroid-pituitary disruption in cancer development in humans is much
less convincing than in animals. Iodide deficiency is associated with increases in
thyroid cancer in some studies but not others. Similarly, an association between
either inborn errors of metabolism affecting thyroid hormone output or
autoimmune-related Graves' disease and cancer is suggested but not proved. It
seems that TSH may at least play some permissive role in carcinogenesis in
humans. Accordingly, one cannot qualitatively reject the animal model; it seems
reasonable that it may serve as an indicator of a potential human thyroid cancer
hazard. However, to the extent that humans are susceptible to the tumor-inducing
effects of thyroid-pituitary disruption, and given that definitive human data are not
available, it would appear that quantitatively humans are less sensitive than rodents
in regard to developing cancer from perturbations in thyroid-pituitary status.
The measured key events and their effects, as well as effects of reversal of the events, are
consistent with what is known about the regulation of thyroid hormone balance, and the
postulated carcinogenic mode of action as summarized above.
Thyroid tumorigenesis, particularly in the male rat, has been observed to be associated
with exposure to a number of pesticides and pharmaceuticals. A pattern of thyroid organ growth,
frequently liver growth, thyroid hormone changes, or changes in hormone metabolism has been
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seen with a large proportion of these compounds. Chemical T effects are parallel to these other
cases.
Thyroid tumors did not appear in the female rats in the 2-year study. Thyrotrophy and
hyperplasia were observed in the females with a 6-month lag after their appearance in the male.
The female is apparently more tolerant of thyroid disruption; whether tumors would have been
seen in the females if the 2-year study had been extended is uncertain.
Relevance to subpopulations
Thyroid hormones are regulated within rather narrow ranges, with normal adult human
serum values often being given as T4--4 to 11 ug/dL and T3--80 to 180 ng/dL. TSH levels
extend over a broader range—0.4 to 8 ug/ml, due to the incorporation in recent years of more
sensitive laboratory methods that have extended the normal range to lower values (Ingbar &
Woeber, 1981; Surks et al., 1990). The upper bound on normal TSH has not changed, and it is
the one of import to considerations of antithyroid effects of chemicals. During development
somewhat higher levels for each of the hormones are noted, with adult hormone values being
reached beyond about 10 years of age (Nicholson and Pesce, 1992). Growth of the thyroid gland
continues for the first 15 years of life, going from about 1 gram at birth to an adult size of about
17 grams (Fisher and Klein, 1981; Larsen, 1982). Early developmental inability to synthesize
adequate thyroid hormone leads to altered physical and mental development (cretinism)
(DiGeorge, 1992; Goldey et al., 1995) and is treatable. The control of normal thyroid growth
during development is not totally known, although the increase in gland size may be independent
of TSH stimulation (Logothetopoulus, 1963). Extended deviations in human thyroid hormone
levels either above or below the normal range are associated with hyperthyroidism and
hypothyroidism, respectively and are treated in the U.S. to restore balance.
Thyroid cancer is a rare condition in the U.S., occurring with an incidence of about
0.004% per year (Greenspan & Strewler, 1997). The incidence is predominantly in persons over
30, and increases in older persons; in children the incidence is at the 1 per million rate. Mortality
rates per 100,000 are above zero only for those older than 35 (Ries et al., 1999).
It is recognized that the human thyroid is susceptible to ionizing radiation, the only
verified human thyroid carcinogen. Children are known to be more sensitive than adults to the
carcinogenic effects of radiation (NRC, 1990; IAEA, 1996). The nature and consequences of
radiation have differences from thyroid disruption by inborn deficits or possible chemical influence
that is not mutagenic. The major effect of ionizing radiation on the thyroid is thought to be due to
mutation. Antithyroid effects can also be induced at elevated radiation doses due to cytotoxicity
of follicular cells with resulting reduction in thyroid hormone and elevation of TSH. Mutagenic
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chemicals, however, do not act totally like radiation: (a) X rays penetrate the body and target
organs without having to be absorbed. Chemicals must be absorbed and distributed to target
organs, (b) Unlike most organic chemicals, radioiodine is actively transported and concentrated in
the thyroid gland, and it becomes incorporated into nascent thyroglobulin. (c) Given that the size
of the thyroid gland is smaller in children than in adults, for a given blood level of radioiodine, the
internal dose to the thyroid of a child is greater than that for an adult, (d) Radioiodine in the
Chernobyl accident was picked up by cattle and incorporated into milk. Due to differences in
milk consumption, the external dose presented to children was greater than to adults.(e) Single
quanta of radiation result in a series of ionizations within biological material, each of which can
react with DNA to induce mutations and affect the carcinogenic process. Chemicals are much less
efficient: they frequently need to be metabolized to active intermediates, with each molecule
interacting singly with DNA, usually by forming adducts which can be converted to mutations, (f)
The spectrum of mutagenic effects vary with the source. Ionizing radiation often results in
deletions and other structural chromosomal aberrations, while chemicals not uncommonly
produce more gene mutations, (g) The thyroid of children is more sensitive to carcinogenic effects
of external radiation on a per unit dose basis than in adults, especially for children less than 5
years of age. Sensitivity decreases with advancing age and seems to disappear in adulthood. It is
estimated that, overall, children may be two or more times more sensitive to carcinogenic effects
of external emitters than are adults (NRC, 1990).
The evidence supports the view that Chemical T's mode of action will not be different for
children. Thyroid cancer is very rare in younger age groups and lower in incidence and mortality
than for older adults. It does not appear that the young have any propensity for thyroid cancer
from which one could infer some underlying cancer process that differs from adults (absent
ionizing radiation treatment or incidents, discussed above). The basic elements of thyroid
function and hormone homeostasis are the same in children and adults with a period of growth
during which children reach lower adult balances. The chemical disruption mode of action of
Chemical T in animals, to the extent that it is applicable to humans, appears equally applicable to
human subpopulations. It is not expected to share the features of radiation.
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D.2.0. EXAMPLE 2: CHEMICAL Z (BLADDER TUMOR)
D.2.1. HAZARD DATA SUMMARY
D.2.1.1. Data Availability
Data include a rat chronic/carcinogenicity feeding study, an 18 month CD-I mouse
carcinogenicity study, a three-generation reproduction study in the rat, and a 2-year feeding study
in dogs. There are no data on the effects in humans of exposure to chemical Z.
A 13-week feeding study in rats included interim sacrifices at 2, 4, and 8 weeks and
establishment of 16-week recovery groups at 8 weeks and a 21-week recovery group at 13
weeks.
D.2.1.2. Tumor Observations
D.2.1.2.1. Tumor Response
D.2.1.2.1.1. Rats. Administration of chemical Z in the diet to male Sprague-Dawley rats at dose
levels of 30,000 ppm or more for 2 years resulted in an increase in bladder urothelial tumors in
male rats. Statistically significant increases (p<0.05) were noted at the high dose only
(40,000/30,000 ppm) in the incidences of transitional cell papillomas, carcinomas, combined
papillomas and carcinomas, and hyperplasia in the 2-year SD rat bioassay (Table D-2). Bladder
calculi were observed in some animals but correlation between stones and tumors was not evident
at final sacrifice.
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Table D-2. Incidence of transitional cell lesions and stones in the bladder
of males from a 2-year SD rat study
Parameter
Dose (ppm)
0
2000
8000
40,000/30,000
N
73
75
78
78
Lesion
Papilloma
1
1
1
5
Carcinoma
2
2
1
16
Combined
3
3
2
21
Hyperplasia
5
7
5
29
Stones
0
0
0
5
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D.2.1.2.1.2. Mice. No increase in tumor incidences was observed in an 18-month bioassay with
mice.
D.2.1.3. Mutagenicity
Chemical Z has not shown mutagenic activity based on results of Salmonella sp. or
micronucleus assays. No evidence exists that the chemical produces effects on DNA synthesis nor
does it appear to be clastogenic. There are no structural alerts suggesting mutagenic potential for
the chemical.
D.2.1.4. Toxicity, Uroliths, and Hyperplasia
There was a strong association among disruptions in urinary physiology, toxicity, uroliths,
and hyperplasia in the 13-week study in mid-dose and high-dose animals (30,000 and 50,000 ppm
respectively, [p<05]). In the control and 8,000 ppm group, no animals had stones and no animals
had hyperplasia (see Table D-3).
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Table D-3. Incidence of bladder hyperplasia and stones in male SD rats
treated up to 13 weeks
Parameter
2 weeks
8 weeks
13 weeks
Dosea
1
2
3
4
1
2
3
4
1
2
3
4
N
10
10
10
10
10
10
10
9
10
10
10
6
Papillary
hyperplasia
0
0
7
8
0
0
9
7
0
0
5
6
Simple
hyperplasia
0
0
2
0
Stones
0
0
3
4
0
0
9
8
0
0
7
6
aDose (ppm): 1 = control, 2 = 8000, 3 = 30,000, 4 = 50,000.
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D.2.1.4.1. Thirteen-Week Study
Urothelial toxicity and disruptions in urinary physiology and urothelial toxicity appeared
early in the study. Early changes in urinary physiology (decreased pH and increased cation
concentration) were observed following 2 weeks of treatment and persisted throughout the
duration of the study. Urothelial toxicity was expressed as edema, cystitis, and hyperplasia;
hyperplasia (simple and papillary transitional cell combined) increased in overall incidence with
continued treatment. It was present in 70% of mid-dose (30,000 ppm) animals and 80% of high-
dose (50,000 ppm) animals following 2 weeks of exposure, and in 70% of the mid-dose group
and 100%) of the high-dose group at 13 weeks. There was some indication of a decrease in
severity of hyperplasia at 13 weeks when compared to earlier time periods, as there was an
apparent shift from the incidence of papillary hyperplasia to simple hyperplasia and a decrease in
the combined incidence of hyperplasia in the 30,000 ppm group of animals.
Uroliths were found to be present as early as 2 weeks (0%, 0%, 30%, and 40%) and the
incidence increased over the period of the study. The incidence of uroliths at termination of the
13-week study was 0%, 0%, 70%, and 100%, but there was a decrease in size and number of
stones per animal at 13 weeks.
D.2.1.4.2. Three-Generation Reproduction Study in Rats
High dose levels (>20,000 ppm in the diet) led to formation of lesions in the urinary tract of
males and females of the Fl, F2, and F3 generations. The lesions included hemorrhage of the
bladder wall, increased pelvic dilation, and papillary necrosis. In the F3 generation, additional
effects noted in renal tissue were hyperplasia of the transitional epithelium and desquamation of
cells in the lumen of the urinary tract. The changes were associated with crystalline or calcareous
deposits.
D.2.1.5. Reversibility of Effects
There was strong evidence of reversibility of bladder stones and bladder hyperplasia. When
animals that had been treated for 8 weeks were returned to basal diet for 16 weeks, uroliths were
found in 30% of 30,000 ppm animals and 25% of high-dose animals. Bladder hyperplasia
(papillary and transitional cell combined) was reduced to 25% and 30% in each of these two dose
groups (Table D-4). An analysis of individual animal data revealed a strong correlation between
the incidence of uroliths and hyperplasia at the termination of the recovery period.
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Table D-4. Reversal of incidence of bladder hyperplasia
and stones following 8 weeks treatment and 16 weeks recovery
Parameter
Dose (ppm)
0
8000
30,000
50,000
N
10
10
10
8
Papillary
hyperplasia
0
0
2
1
Simple
hyperplasia
0
0
1
1
Stones
0
0
3
2
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1 D.2.1.6. Blood and Urine Chemistry
2 Chemical Z administration resulted in increases in blood phosphorus and carbon dioxide
3 (data not shown). Urinalyses (Table D-5) showed elevated calcium levels, reduced urinary
4 phosphorus, and a profound lowering of urinary pH (5.0), which began at 2 weeks and persisted
5 throughout the 13-week study in the 30,000 and 50,000 ppm group of rats. These changes
6 occurred in the presence of bladder stones, which were reported to consist of 33% calcium and
7 23% phosphorus.
8
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Table D-5. Clinical chemistry values (urine) in male SD rats treated up to 13 weeks
Parameter
2 weeks
8 weeks
13 weeks
Dose
1
2
3
4
1
2
3
4
1
2
3
4
N
10
10
10
10
10
10
10
9
10
10
10
6
Calcium -
mg/dL
6
11
56b
36c
11
11
18
65b
5
7
14b
58b
Phosphorus
- mg/dL
90
62
2b
13c
109
90
19
lb
57
67
26
lb
pH
7
6.5
5b
5b
7.4
6.9
5.8b
5.0b
7.
2
6.
7
6.0b
5.0b
Stones
0
0
3
4
0
0
9
8
0
0
7
6
aDose (ppm): 1 = control, 2 = 8000, 3 = 30,000, 4 = 50,000.
V-01-
cp< 05
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D.2.1.7. Metabolism
Upon ingestion by rats, the ethyl moiety of chemical Z is rapidly absorbed, hydrolyzed to a
phosphite, and oxidized via acetaldehyde and acetate to carbon dioxide and water. Absorption of
the phosphite moiety leads to increased blood phosphorus levels. There is also an increase in
blood calcium load, which leads to increased excretion of calcium via the urine. Ethyl phosphite
moieties and carbon dioxide are also eliminated via the urine. A marked depression of urinary pH
(5.0) results from acidification of the urine by carbon dioxide. An aluminum moiety of the parent
chemical is poorly absorbed, and most is eliminated in the feces. The phosphite metabolite, the
major urinary metabolite, was not shown to express carcinogenic potential when administered to
Sprague-Dawley rats at dose levels up to 32,000 ppm. It also does not express any mutagenic
potential and does not have any structural alerts.
D.2.1.8. Structure-Activity Relationships
There are no data on structurally related chemicals.
D.2.2. MODE-OF-ACTION ANALYSIS
D.2.2.1. Summary Description of Postulated Mode of Action
Chemical Z produces transitional cell tumors in male Sprague-Dawley rats. The mode of
action includes disruption in urinary physiology, including precipitation of calcium and
phosphorus and formation of bladder calculi. The stones irritate the urothelium of the bladder,
followed by transitional cell hyperplasia and bladder tumor formation. Disruption of urinary
physiology is a consequence of a metabolic sequence involving (1) absorption and metabolism of
the ethyl moiety to carbon dioxide, resulting in a reduction in urinary pH; and (2) absorbtion of
the phosphite moiety, which leads to increased blood phosphorus levels and increased release of
calcium into the urine. Increases in water consumption followed by increased urinary volume may
contribute to bladder toxicity, but a precise role of increased urinary volume has not been
established.
The mode of action for chemical Z is consistent with other data that demonstrate that solid
masses in the rodent bladder, regardless of their origin—insertion of solid materials, including inert
pellets, precipitation of administered chemicals (e.g., melamine) or disruption of urinary
physiology (e.g., diethylene glycol)—lead to urothelial toxicity and the formation of tumors.
D.2.2.2. Key Events
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The key precursor events associated with bladder tumor formation following administration
of chemical Z to rats include increased blood phosphorus and carbon dioxide, elevated urinary
calcium and volume, decreased urinary pH and phosphorus, formation of bladder stones, and
irritation and hyperplasia of the urothelium.
D.2.2.3. Strength, Consistency, and Specificity of Association of Tumor Response with Key
Events
The only tumor response seen in animal studies is bladder tumors in male Sprague-Dawley
rats. Studies in dogs and mice showed no effect on the bladder. The rat tumor response was seen
only at high doses that lead to key precursor effects: altered urinary physiology (volume, calcium,
pH) results in stones and produces toxicity and hyperplasia of the urothelium. The high-dose
changes were noted in a rat chronic, a rat subchronic, and a three-generation reproduction study
in rats. The key events, including hyperplasia, were observed to be reversible in subchronic
stop/recovery studies. Administration of the major metabolite of chemical Z, monosodium
phosphite, fails to reduce urinary pH, increase urinary volume, or produce nonneoplastic or
neoplastic lesions of the bladder. The database on chemical Z is sufficient to evaluate the
proposed mode of action despite the absence of more complete information on the composition of
the stones and questions regarding the absence of toxicity following the administration of
monosodium phosphite. There is a high degree of confidence that the findings accurately reflect
the effects associated with administration of the chemical. No data gaps were identified that
would substantially alter the evaluation of the proposed mode of action.
D.2.2.4. Dose-Response (D/R) Relationships
The 2-year bioassay showed urothelial hyperplasia, transitional cell papillomas, and
transitional cell carcinomas and a few bladder stones at 40,000/30,000 ppm. Of 78 high-dose
animals, 37% showed bladder tumors. Tumors, hyperplasia, and stones were not increased at
8000 ppm. A special 13-week feeding study demonstrated that key events—increased urinary
calcium levels, decreased urinary phosphorus levels, decreased pH, bladder stones, irritation,
edema, and hyperplasia—occurred consistently only at dose levels of 30,000 ppm or greater. A
strong dose-response correlation was shown between calculus formation and hypercalciuria,
acidic urine, and bladder hyperplasia. In a rat reproduction study, bladder effects were noted at
24,000 ppm but not at 12,000 ppm.
D.2.2.5. Temporal Association
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A subchronic rat study with serial sacrifices at 2, 4, 8, and 13 weeks, including evaluation
of 16-week recovery groups after 8 weeks and a 21-week recovery group after 13 weeks, was
performed. By 2 weeks of administration, chemical Z produced stones that filled the bladder and
resulted in advanced papillary hyperplasia. The number and size of stones was greatest at two
weeks and there was a progressive decrease over the 13 week period. Early changes in urinary
physiology (decreased urinary pH, increased calcium concentration, and decreased phosphorus
concentration) were observed following 2 weeks of treatment and persisted throughout the
duration of the study. Observation of the 8-week treatment/16-week recovery groups showed
that incidence of both stones and hyperplasia significantly decreased as compared with incidence
in animals sacrificed at 8 weeks. Also, upon cessation of dosing at 13 weeks, the incidence of
animals with stones, the incidence of papillary hyperplasia, and the severity of hyperplasia
decreased significantly by the end of a 21-week recovery period (data not shown). The changes
noted within 2 weeks of dosing appear to have set in motion a series of events beginning with
increased urinary calcium concentrations, followed or accompanied by stone formation, irritation
of the bladder urothelium, hyperplasia and, eventually, neoplasia.
D.2.2.6. Biological Plausibility and Coherence of the Database
Long-term and subchronic studies with chemical Z have demonstrated a dose correlation
between development of stones and bladder tumor formation in male rats. Data from the 13-
week study indicate a rapid onset of effects (changes in urinary parameters, formation of stones,
and hyperplasia within 2 weeks of dosing) and adaptation of treated animals to chemical Z
exposure by 13 weeks (decreased numbers and size of stones per animal, decreased severity of
hyperplasia). Tumors were observed only at doses at which key events were observed.
Additional bioassay data provide support for the association of tumors in rats with the key
events in rats and the absence of both tumors and similar key events in other species treated with
chemical Z. Treatment of rats in a three-generation reproduction study at high dose levels
(>20,000 ppm in the diet) led to formation of lesions in the urinary tract of males and females.
When administered to dogs at dose levels up to 40,000 ppm in the diet for up to 2 years, the
chemical produced minimal toxic effects overall, no effects on the urinary tract, and no tumors.
Chemical Z produced no effects in mice when administered up to a dose level of 20,000/30,000
ppm in the diet for 2 years.
Observations with chemical Z are in keeping with those observed in many other
experimental settings. Stones, regardless of their chemical makeup, are irritating to the rodent
bladder, causing irritation, hyperplasia, and eventually neoplasia.
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There are some uncertainties regarding the role of certain findings following chemical Z
administration. Generally, an increase in urinary pH is associated with the precipitation of calcium
and phosphorus-containing stones in rats. However, stones are formed in the presence of a low
urinary pH in rats administered chemical Z. It is also unclear whether or not the acidic
environment of the urine (most likely a consequence of the conversion of the ethyl moiety to
carbon dioxide in the blood) contributes to or enhances any effects noted in bladder tissue in rats.
There was a paucity of stones in high-dose animals at termination of the 2-year study but a higher
incidence of bladder tumors, which suggests that bladder stones may not be the causative factor
involved in bladder tumor formation. Other considerations discount this presumption. First, a
number of the high-dose animals showed hydronephrosis or dilation of the ureters, presumptive
indications of past urinary tract obstruction. Second, the 13-week study provided evidence that
bladder calculi develop rapidly (within 2 weeks), but then decreased in frequency and size. The
decrease in size and number of bladder calculi was accompanied by a decrease in severity of
bladder hyperplasia in animals treated with 30,000 ppm of chemical Z. Third, it is recognized that
a constant ppm of an agent in the diet results in a reduction in dose per unit body weight as an
animal grows. Finally, the increased urinary volume or decreased urinary pH may have led to a
dissolution of stones over time.
The absence of bladder stones and urothelial toxicity following administration of the major
metabolite, monosodium phosphite, is puzzling, as one might expect administration to rats would
lead to similar bladder effects as with chemical Z. However, the metabolite when administered to
rats, leads to an increase in blood levels of phosphorus but does not alter urinary volume or pH as
would be expected with an increase in sodium consumption. Considering the high dose-level of
metabolite administered to rats (32,000 ppm), it is unlikely an additional bioassay using higher
dose-levels would provide useful information.
D.2.2.7. Other Modes of Action
Chemical Z is not mutagenic in short-term tests and it does not have a structure
suggesting biological reactivity. No other modes of action, apart from that postulated, are in
evidence. The fact that bladder tumors were the sole tumors seen in rats and that no other species
showed tumors or other toxicities like those in the rat make it less likely that the agent has another
generalized mode of action.
D.2.2.8. Conclusion
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The available bioassay data on chemical Z are sufficient to support the postulated mode of
action that the chemical, which lacks mutagenic potential, leads to bladder tumor formation in
male rats through a sequence of key events involving perturbations in urinary physiology,
especially increased calcium concentration, calculus formation, urothelial irritation, hyperplasia,
and neoplasia.
D.2.3. RELEVANCE OF THE MODE OF ACTION TO HUMANS
Bacterial infection, urinary stones or a combination of the two may be risk factors for
human urinary tract cancer (Burin et al., 1995; Davis et al., 1984; Gonzalez et al., 1991; Kawai et
al., 1994; Hiatt et al., 1982). Infection of the bladder with Schistosoma haematobium leads to
bladder tumors, and part of its action may be associated with stone formation (IARC, 1995). A
significant relationship has also been shown between spinal cord injury and bladder cancer;
chronic infection and stones are found in individuals so affected (Bickel et al., 1991; Broecker et
al., 1981; Dolin et al., 1994; El-Marsi and Fellows, 1981; Stonehill et al., 1996). Case control
epidemiologic studies (relative risks less than three) suggest associations between bladder cancer
and urinary tract stones (Burin et al., 1995; Gonzalez et al. 1991). A large cohort study supports
the association shown between bladder stones and bladder cancer (Chow et al., 1991). Taken as a
whole, stones may play some role (particularly, along with infection) in bladder cancer formation.
Bladder cancer is a disease of advancing age, with about 2/3 of all cases occurring among persons
aged 65 years or older (Hankey et al., 1993).
Stones occur much more frequently in the upper urinary tract than in the bladder of
humans (about 10% of urinary stones are found in the bladder), presumably because the upright
posture of humans predisposes them to expelling stones through the urethra once a stone passes
from the kidney to the bladder (Hiatt et al., 1982; Johnson et al. 1979; DeSesso, 1995). This
characteristic, as well as the pain which accompanies such stones and leads to their surgical
removal. Stones in the rodent bladder tend to be retained, because of their horizontal position.
These findings suggest suggest that there may be a lower susceptibility of humans compared to
rodents to the development of urinary tract tumors associated with stones.
Precipitation of chemicals in the urinary tract with the formation of stones is a common
finding, with about 12% of males and 5% of females having a history over a lifetime of at least
one stone (Johnson et al., 1979). Compared to adults, urinary stone formation in children is an
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uncommon occurrence except in individuals with a predisposing condition, such as, various inborn
errors of metabolism (e.g., cystinuria) and congenital malformations (Gearhart et al., 1991). The
prevalence of urinary stones in children is about 1 case per 20,000 per year (0.005%) (Khoory et
al., 1998). Only about 5% of stones are initially manifest during the first 20 years of life (Johnson
et al., 1979). Causes of urinary stones in children are remarkably similar to those of adults
(Khoory et al., 1998; Stapleton, 1996). Like with adults, the urine of children varies in pH and
osmolality, particularly in response to diet and physiologic stressors (e.g., exercise, heat). Urinary
excretion of chemicals occurs throughout life, although there may be quantitative differences
associated with a number of factors including disease states and nutritional status. Stones used to
be more common in children in developed countries than they are now, largely due to
malnutrition, which is still a problem in developing nations today (Trinchieri, 1996).
Chemical Z is converted to metabolic derivatives through simple hydrolysis, a chemical
conversion that does not depend on enzymatic activity. It is not plausible that differences in levels
of enzymatic activity, such as detoxification via hepatic metabolism or metabolism in other tissues
will alter, qualitatively, responses in population subgroups such as the aged, the infirm, or infants
and children who may be exposed to Chemical Z.
In summary, the potential human carcinogenic hazard of the chemical cannot be dismissed
for Chemical Z. Chemical Z poses a carcinogenic hazard to humans only under conditions that
would lead to the formation of bladder stones. It is reasonable to conclude that the mode of
action involving stone formation for Chemical Z that has been developed for adult animals may be
applicable to young animals and to children. Information suggests that effects in the young may
not be any greater than in adults and, in fact, the young may be less susceptible unless there are
rare extenuating factors.
3.0. EXAMPLE 3: CHEMICAL D
D.3.1. HAZARD DATA SUMMARY
D.3.1.1. Data Availability
Human data are inadequate to establish a basis for carcinogenicity. Experimental data
include:
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• Three chronic toxicity and carcinogenicity studies in rats and mice: an inhalation study,
an oral dietary study, and an oral gavage study;
• Subchronic studies by the oral and inhalation routes in rats and mice;
• Inhalation developmental toxicity studies in rats and rabbits;
• An inhalation two-generation reproductive toxicity study in the rat;
• In vitro and in vivo genotoxicity studies;
• Toxicokinetic and metabolism studies; and
• Protein binding studies.
D.3.1.2. Carcinogenicity/Chronic Toxicity
Chemical D has been shown to cause increased tumor incidences in rats and mice. The
tumor responses seem to be dependent on the tested animal species, sex, dose, and route of
administration. Results of available chronic bioassays are summarized in Table D-6.
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Table D-6. Summary results of chronic bioassays
Study/dose
F344 rats
B6C3F1 mice
Oral gavage
Rat study. 0, 25, 50 mg/kg
(5 d/wk for 2 yr)
Mouse study. 0, 50, 100
mg/kg (5 d/wk for 2 yr)
Forestomach:
Papillomas (males: 1/50,
2/50, 8/50; females: 0/50,
2/50, 3/50)
Carcinomas (males only-
0/50, 0,50, 4/50)
Basal cell and epithelial
hyperplasia (dose-related;
males and females)
Liver:
Adenomas (males: 1/50, 6/50,
7/50)
Carcinomas (males: 0/50,
1/50, 3/50)
Forestomach:
Papillomas (males: 0/50,
1/50, 5/49; females: 0/50,
2/50, 7/50)
Carcinomas (females only:
0/50,1/50, 4/50)
Basal cell and epithelial
hyperplasia (dose-related;
males and females)
Lung:
Adenomas (males: 2/50, 4/50,
8/49; females: 2/50, 4/50,
7/50)
Carcinomas (males only:
0/52, 2/52, 4/49)
Oral dietary
Rat study. 0, 2.5, 12.5, 25
mg/kg/day for 2 yr
Mouse study. 0, 2.5. 25, 50
mg/kg/day for 2 yr
Forestomach:
Basal cell and epithelial
hyperplasia (dose-related;
males and females)
Liver:
Adenomas (significant in
males only: 2/50, 1/50, 6/50,
9/50)
No histopathologic changes
Inhalation
Rat study. 0, 5, 20, 60 ppm (6
hr/d 5 d/wk for 2yr)
Mouse study. 0, 5, 20, 60
ppm (6 hr/d 5 d/wk for 2yr)
Nasal cavity:
Epithelial hyperplasia (dose-
related; males and females)
Nasal cavity:
Epithelial hyperplasia (dose-
related; males and females)
Lung:
Adenomas (males only: 2/50,
3/50, 6/50)
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• In rats, chemical D caused dose-related increases in liver tumors (males only) and
forestomach tumors (both sexes) via oral gavage, but only liver tumors (males high
dose only) by ingestion. No tumors were found in an inhalation study.
• In mice, chemical D caused dose-related increases in forestomach and lung tumors
(both sexes) by oral gavage, but no tumors were observed in the oral dietary study.
Chemical D only induced an increased incidence of lung tumors in male mice exposed
to the high dose by inhalation.
• Nonneoplastic changes were observed in the forestomach of treated rats (gavage and
dietary studies) and mice (gavage only) of both sexes. Chemical D also induced
nonneoplastic changes in the nasal mucosa of rats and mice of both sexes via
inhalation.
D.3.1.3. Subchronic Toxicity
Subchronic toxicity studies have been conducted in rats and mice by the oral and
inhalation routes. The primary organs affected were the forestomach (rats) and the liver (mice) via
oral exposure, and the nasal cavity and respiratory tract of both rodent species via inhalation.
D.3.1.3.1. Oral Studies
Groups of F344 rats (10 animals of each sex per dose group) were administered 0, 5, 15,
50, or 100 mg/kg/day of chemical D via their diets for 13 weeks. Dose-related decreases in body
weight gain were observed in treated males and females. Basal cell hyperplasia and hyperkeratosis
of the forestomach was found in males and females rats treated with chemical D at the three
highest doses.
B6C3F1 mice (10 animals of each sex per dose group) were administered 0, 25, 50, 100,
or 175 mg/kg/day via their diets for 13 weeks. Body weight gains of treated males and females
were depressed in a dose-related manner compared to controls. Histologic changes were noted in
the liver and were characterized as decreased hepatocyte size in all treatment groups. This
observation was consistent with decreased hepatocellular cytoplasmic glycogen.
D.3.1.3.2. Inhalation Studies
F344 rats (10 animals of each sex per dose group) were exposed to 0, 10, 30, 90, or 150
ppm of chemical D for 6 hr/day, 5 days/week for 13 weeks. Treatment-related effects included
depressed body weight gain (at 30 ppm and greater), degenerative changes in nasal olfactory
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epithelium, and hyperplasia of respiratory epithelium in both males and females (at 90 and 150
ppm).
B6C3F1 mice (10 animals of each sex per dose group) were exposed to 0, 10, 30, 90, or
150 ppm of chemical D for 6 hr/day, 5 days/week for 13 weeks. Treatment-related effects
included depressed body weight gain (at 30 ppm and greater), and histopathologic changes in the
respiratory and olfactory epithelium of the nasal mucosa of both sexes exposed to 30, 90, and 150
ppm).
D.3.1.4. Developmental and Reproductive Toxicity
Pregnant F344 rats and New Zealand White rabbits were exposed to 0, 20, 60, or 120
ppm of chemical D during gestation days 6-15 (rats) and 6-18 (rabbits). Maternal effects
(decrease body weight gain) were observed in rabbits and rats, in all treatment groups. A slight
but statistically significant increase in the incidence of delayed ossification of the vertebral centra
was observed in rats exposed to the high dose level. No developmental effects were observed in
the rabbit study.
Exposure of F344 rats to 0, 10, 30, or 90 ppm of chemical D for up to two generations
did not induce any effects on reproductive parameters or neonatal growth and survival in any of
the generations. Parental effects were limited to epithelial degeneration of the nasal mucosa of the
adults rats exposed to 90 ppm.
D.3.1.5. Mutagenicity
Chemical D was tested in many assays for gene mutation and chromosomal aberrations, as
well as assays indicative of DNA damage, DNA strand breaks, and DNA alkylation. A
heterogeneous database is found (a few in vitro positive responses and several negative results).
It has been suggested that this heterogeneity is due to different studies that have used different
test materials containing varying levels of impurities.
A few studies demonstrated that chemical D was weakly positive in the Ames bacterial
assays in the presence of liver microsomes. Addition of cytosolic enzymes, presumably containing
the detoxification enzyme glutathione transferase (GST), abolished mutagenic activity. Studies
for chromosomal aberrations in vitro assays using mammalian cells have tended to be negative.
There are a few positive results reported, but these are inconsistent with negative studies
conducted in the same assay.
There are very few in vivo genotoxicity studies on chemical D. Chemical D has been
found to be negative in a mouse micronucleus assay when tested up to oral doses of 175 mg/kg.
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Chemical D has been reported to produce sister chromatid exchanges (SCEs) in mice. It should
be noted that this assay has a low specificity for predicting carcinogenesis (i.e., a high rate of false
positives compared to results of the rodent cancer bioassay). No dominant lethal effects (i.e.,
germ cell genetic damage) were found in rats exposed to chemical D by inhalation up to 150 ppm.
In vivo DNA binding studies were conducted in rats and mice. Rats were exposed acutely
to chemical D at doses of 0, 10, 25, or 100 mg/kg. Mice were exposed acutely by inhalation to
chemical D at 0, 30, and 60 ppm. No significant DNA binding (as measured by 32P postlabeling
assay) was seen in liver tissue from treated rats and lung tissue from exposed mice. In the mouse,
DNA strand breakage was also studied by alkaline elution. Negative results were reported.
D.3.1.6. Toxicokinetic and Metabolism Studies
Toxicokinetic and metabolism studies in rats and mice have demonstrated that chemical D
was rapidly absorbed by the oral and inhalation route. Blood half-lives were less than 10 minutes.
Mercapturic acid conjugate of chemical D was the only major metabolite detected in the urine of
treated rats and mice (about 80-90% of administered dose). Conjugated metabolites of chemical
D epoxide were not detected in the urine of treated rats and mice.
Significant dose-related decreases in hepatic and lung tissues of GSH were observed in
rats treated acutely with chemical D at oral doses of 0, 5, 20, 50, or 100 mg/kg, and in mice
exposed acutely by inhalation to 0, 30, 60, or 150 ppm, respectively.
D.3.1.7. Protein Binding Studies
Chemical D was found to bind with tissue proteins in the forestomach and liver of rats
treated acutely with oral doses 10, 50, and 100 mg/kg. Chemical D binding to tissue proteins was
also found in the lung of mice exposed via acute inhalation at 30, 60, or 100 ppm.
D.3.2. MODE-OF-ACTION ANALYSIS
D.3.2.1. Summary Description of Postulated Mode of Action
It is postulated that chemical D causes tumors in rats and mice only when it is
administered at high doses and/or by bolus administration that overwhelms the detoxifying
mechanisms. The tumorigenic responses also appear to be closely associated with tissue toxicity
(e.g., rat and mouse forestomach) and high background spontaneous tumors (e.g., mouse lung, rat
liver). These observations, coupled with the lack of significant in vivo mutagenic activity, lead to
the postulation that chemical D-induced tumorigenicity is likely to be operated by a nonmutagenic
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mode of action, and appears to be secondary to toxicity and reparative cell proliferation. At high
doses, a mutagenic mode of action may also be involved.
It is postulated that once absorbed, chemical D is biotransformed spontaneously or by
microsomal mixed functional oxidases (MFO) to an epoxide derivative that can react directly with
DNA. Both parent chemical D and the epoxide derivative are rapidly conjugated with glutathione
(GSH), which then can be excreted in the urine, mainly as the mercapturic acid conjugate of
chemical D. Under normal physiologic conditions, i.e., at nonsaturating doses, chemical D is
effectively detoxified as glutathione conjugate, and epoxidation does not take place in any great
extent. At high doses, chemical D is expected to react chemically with thiols in proteins, causing
tissue toxicity (forestomach), depleting tissue GSH, and causing proliferation of high background
spontaneous foci of altered cells (rat liver and mouse lung) leading to tumorigenesis. As less GSH
is available for detoxifying chemical D, more chemical D is metabolized to the mutagenic epoxide
derivative, which may play a role in the carcinogenic process.
D.3.2.2. Key Events
D.3.2.2.1. Metabolism
It is hypothesized that epoxidation of chemical D does not take place to any great extent
since conjugated metabolite(s) of chemical D epoxide have not been detected in the urine of
treated rats and mice. This finding was based only on acute exposure to chemical D. The
metabolic profile of chemical D might differ under repeated exposures, particularly because
chemical D has been found to deplete tissue GSH. Additional in vitro and in vivo metabolism
studies are needed to further elucidate the potential role of MFO and epoxidation of chemical D.
D.3.2.2.2. Tissue Toxicity
It is postulated that chemical D-induced tumorigenicity is secondary to toxicity. The only
target organ that exhibits both toxicity and tumorigenicity is the forestomach of rats and mice.
Liver and lung toxicities have not been observed in chronic studies, although they have been
reported in subchronic studies at higher doses. On the other hand, nasal toxicity was observed in
exposed rats and mice, but no tumors were found.
Furthermore, the data supporting the postulated mechanism(s) of chemical D-induced
toxicity are limited. It is hypothesized to be mediated by chemical D binding to tissue proteins.
The only available information is the finding from acute oral and inhalation studies showing dose-
related chemical D binding to proteins of the liver and forestomach of rats, and lung of mice,
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respectively. Additional studies are needed to investigate the potential toxicity of chemical D at
the biochemical, molecular, cellular, and tissue levels.
D.3.2.2.3. Depletion of GSH
The ability of chemical D to deplete tissue GSH has been demonstrated to take place only
in the liver and forestomach of rats following acute ingestion and in the lung of mice via acute
inhalation. Additional data are needed to examine the effects of chemical D on GSH levels in
target organs as well as unaffected organs after repeated exposure.
D.3.2.2.4. Proliferation Activity
There is no information to substantiate the postulate that chemical D promotes highly
spontaneous rat liver or mouse lung altered cells. Cell proliferation and mutation spectra studies
are needed to examine the proliferative potential of chemical D.
D.3.2.3. Strength, Consistency, Specificity of Association of Tumor Response With Key
Events
As discussed above, the postulated key events have not been clearly established. Thus, it is
difficult to determine how well these key events relate to the observed tumorigenic responses. In
general, the relationship between toxic and carcinogenic effects of chemical D on the forestomach
of rats and mice is relatively stronger and more consistent than its effects on the rat liver and the
mouse lung.
D.3.2.3.1. Forestomach Tumors
Subchronic studies and chronic studies in rats and mice demonstrate that the forestomach
is the primary target by oral exposure to chemical D. The rat appears to be more susceptible to
chemical D-induced forestomach toxicity than the mouse.
Dose-related neoplastic and nonneoplastic lesions of the forestomach were observed in
treated rats and mice of both sexes when chemical D was administered by gavage. In contrast,
only hyperplastic lesions of the forestomach were found in male and female rats following
subchronic and chronic dietary exposures to chemical D. No histopathologic changes were
observed in the forestomach of treated mice in the subchronic and chronic dietary studies.
D.3.2.3.2. Liver Tumors
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Chronic exposure of chemical D caused increased incidences of hepatic adenomas in male
rats when administered in the diet and by gavage. However, nonneoplastic changes in the liver
were not observed in male rats after chronic or subchronic oral exposure to chemical D.
D.3.2.3.3. Lung Tumors
Chemical D induced increased incidences of lung adenomas in exposed mice via chronic
inhalation (males only) and oral gavage. Nonneoplastic changes in the lung of exposed mice were
not reported in the chronic study.
D.3.2.4. Dose-Response Relationships
As discussed above, dose correlations were demonstrated for chemical D-induced toxicity
and/or carcinogenicity in the various target tissues of treated rats and mice. Dose-related
depletion of tissue GSH was demonstrated with chemical D. However, no dose-related data are
available for other toxicokinetic and metabolic parameters (absorption, uptake, distribution,
metabolism, clearance and excretion of chemical D and metabolites), in vivo DNA binding, and
other key events (e.g., cytotoxicity, cell proliferation) that are postulated to be involved in the
tumorigenic process.
D.3.2.5. Temporal Association
While there are limited data indicating an association between chemical D-induced
carcinogenicity and related toxicity (mostly for the forestomach), there are no data to discern the
temporal association of these effects. Moreover, no data are available to establish the sequence of
key events at the biochemical, molecular, or cellular levels that might mediate the tumorigenic
responses.
D.3.2.6. Biological Plausibility and Coherence of the Database
The postulated mode of action for chemical D-induced forestomach tumors in rats and
mice appears plausible and coherent with current knowledge. Many chemicals that are strong
irritants have been shown to cause forestomach tumors via bolus administration. Similarly, the
mouse lung appears to be more susceptible to the carcinogenic actions of many toxicants by
inhalation. On the other hand, the observation that chemical D induces liver tumors only in the rat
is not consistent with the general observation that the mouse is more susceptible than the rat to
the carcinogenic effects of many halogenated hydrocarbons.
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D.3.2.7. Other Modes of Action
Chemical D bears a structural resemblance to several short-chain halogenated
hydrocarbons that are also animal carcinogens. Chemical D is expected to generate a mutagenic
epoxide. Chemical D has been shown to exhibit weak mutagenic responses in a number of in vitro
bacterial assays in the presence of liver microsomes, although addition of cytosolic enzymes,
presumably containing GST, has been shown to abolish the mutagenic activity. Several
cytogenetic assays demonstrated that chemical D can cause chromosomal damage in mammalian
cells. Thus, a mutagenic mode of action cannot be entirely ruled out for chemical D.
D.3.2.8. Conclusion
There is little evidence to support a conclusion that chemical D-induced tumorigenicity in
rats and mice is mediated by a nonlinear mode of action. The key events responsible for the
tumorigenic responses are not well defined and a temporal association of these key events has not
been fully investigated. Furthermore, it is still not possible to rule out a mutagenic mode of action
by chemical D. Additional data on the chemical interactions of chemical D with macromolecules,
and the nature of cytotoxic insults in target tissues and their relationship to tumor formation are
needed.
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APPENDIX E. NONLINEAR DOSE-RESPONSE:
MARGIN OF EXPOSURE ANALYSIS
1 [To Be Developed]
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APPENDIX F. DOSE-RESPONSE ASSESSMENT FOR A CARCINOGEN POSING
HIGHER RISKS AFTER CHILDHOOD EXPOSURE
a. Introduction
Compound K is a carcinogenic to humans by all exposure routes. This conclusion is based
on: (1) consistent epidemiologic evidence of a causal association between occupational exposure
and the development of angiosarcoma, an extremely rare tumor; (2) suggestive epidemiological
evidence that cancers of the brain, lung, and lymphopoietic system are associated with exposure;
(3) consistent evidence of carcinogenicity in rats, mice, and hamsters via the oral and inhalation
routes; (4) mutagenicity and DNA adduct formation by compound K and its metabolites in
numerous in vivo and in vitro test systems; and (5) efficient absorption via all routes of exposure
tested, followed by rapid distribution throughout the body.
Carcinogenicity involves genetic toxicity and is understood in some detail. Compound K
is metabolized to a reactive metabolite, probably an epoxide, which is believed to be the ultimate
carcinogenic metabolite. The reactive metabolite then binds to DNA, forming DNA adducts that,
if not repaired, ultimately lead to mutations and tumor formation. Therefore, a linear
extrapolation was used in the dose-response assessment. Because of uncertainty regarding
exposure levels in the occupationally exposed cohorts, an inhalation unit risk of 2xl0"6 per ug/m3
was based on chronic inhalation studies in rats (not presented here).
Evidence has also been reported indicating increased sensitivity to early-life exposure.
This case study shows how to use such evidence in a quantitative risk assessment. To focus on
early-life exposures, the hazard assessment and dose-response assessment for chronic exposure
(including derivation of the inhalation unit risk of 2x10"6 per ug/m3) are not presented here.
b. Dose-response data for early-life exposure
A dose-rate study compared responses to different dosing regimens, in which rats inhaled
compound K for 100 hours, starting at 13 weeks of age or 1 day of age (see table F-l). No effect
was observed for 100-hr exposures starting at 13 weeks, but 100-hr exposure starting at 1 day
had a clear carcinogenic effect, causing both angiosarcomas and hepatomas.
Tumor incidences in the newborn rats were also compared with rats inhaling compound K
for 52 weeks starting later in life (at 13 weeks) (see table F-2). Angiosarcoma incidence was
comparable from 52-week exposure starting at 13 weeks and 5-week exposure starting at 1 day.
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Hepatoma incidence, however, was high after newborn exposure but virtually absent after chronic
exposure starting later in life.
These data illustrate two phenomena that indicate higher cancer risks from childhood
exposure: (1) high incidence of a tumor (angiosarcomas) also caused by adult exposure, and
(2) occurrence of another tumor (hepatomas) not associated with adult exposure. The data
suggest that risks from short-term, early-life exposure may not be reversible even in the absence
of further exposure. The data do not, however, help us understand why early-life exposure poses
greater risks. It could be that the metabolized dose is higher in newborns than in adults (either
through more efficient metabolism, slower elimination, or a higher saturation point), alternatively,
metabolized doses could be comparable in newborns and adults, but newborns could be
biologically more sensitive to the same dose. Without understanding the mode of action early in
life, we can nonetheless use these data to estimate the higher cancer risks caused by early-life
exposure.
c. Dose conversion
Extensive pharmacokinetic studies show that the carcinogenic effects are caused by a
metabolite and that metabolism becomes saturated below the tested doses. A PBPK model was
fitted and validated (using independent data) to convert the experimental inhaled concentrations
to equivalent human concentrations (see table F-3). This involved two steps: (1) convert
experimental concentrations in air (ppm) to tissue concentrations in rat liver (mg metabolite per
L liver), and (2) convert these tissue concentrations to equivalent human concentrations in air
(ppm or mg/m3). The inhalation unit risk for chronic adult exposure was derived using doses
from this model.
Although the PBPK model was fitted using data on mature rats and adult human males,
dose estimates from this model were also used for dose-response modeling of tumors from early-
life exposure. Similarly, although liver tissue concentrations were used as the dose metric in the
PBPK model, this model was also used for angiosarcomas and angiomas at all sites (NTP
guidance indicates that these tumors should be combined). Although the ideal would be to have
pharmacokinetic information on various tissue concentrations in children, these studies have not
been conducted. The lack of this information introduces some uncertainty into the results. Use of
the PBPK model reflects a conscious decision that a credible dose-response model would be
based on saturable metabolism and not on administered concentrations alone.
Although it is standard practice to calculate lifetime average daily doses for carcinogens
(U.S. EPA, 1992), a different approach may be appropriate when considering effects of childhood
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exposures if children are more sensitive than adults. Specifically, it may not be appropriate to
average childhood exposures over a full lifetime, since that implies that childhood exposure is
equivalent to full-life exposure at a lower rate. Consequently, the dose estimates from the PBPK
model are not averaged over a lifetime. Instead, the average dose during the early-life period (in
this experiment, 5 weeks) is used. That is, the administered concentration is reduced to reflect
intermittent exposure of 4 hr/d, 5 d/wk, but there is no further reduction by the ratio of the early-
life period (5 wk) to a lifetime. This childhood exposure estimate is applied to the childhood-
specific unit risk estimate calculated below. (If a unit risk estimate could not be calculated from
the early-life experiments and the adult unit risk estimate were used instead, the adult unit risk
would be adjusted for children as discussed in section 3.5.2.)
d. Analysis in the range of observation
In the range of observation, incidences of angiosarcomas or hepatomas (from table F-l)
are modeled separately as functions of equivalent human concentration based on metabolized dose
(from table F-3) using a quantal polynomial model of the form
p(d) = 1 - exp(-q^-... - qjt), qx, . . . , qk> 0
The resulting points of departure are LEC10 = 36 ppm for angiosarcomas and LEC10 = 33 ppm for
hepatomas. Converting these to units of ug/m3 (for this compound, 1 ppm = 2600 ug/m3) yields
LEC10 = 9.4xl04 ug/m3 for angiosarcomas and LEC10 = 8.6xl04 ug/m3 for hepatomas.
e. Extrapolation to lower doses
The available mechanistic information, which indicates a reactive metabolite that binds to
DNA and forms DNA adducts that ultimately lead to mutations and tumor formation, supports
linear extrapolation to lower doses. Linear extrapolation follows the line from the point of
departure to the origin (zero dose, zero excess risk). The slope of this line is 0.10/LEC10.
Accordingly, the unit risk estimates are l.lxlO"6 per ug/m3 for angiosarcomas and 1.2xl0"6 per
ug/m3 for hepatomas.
f. Combining unit risk estimates for multiple tumor types
To obtain an estimate of overall cancer risk, the unit risks for the induced tumor types are
combined. In the absence of individual animal pathology data, a neutral assumption is that the
tumor types are independent. In this case, the induction of angiosarcomas but not hepatomas by
later-life exposure suggests that these tumor types are caused by different modes of action and
may be independent. Under an assumption of independence, the combined unit risk is
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1.lxl0"6 + 1.2xl0"6 - (l.lxlO"6 x 1.2xl0"6) = 2.3xl0"6 per ug/m3
g. Strengths and limitations of the data
Although the data on newborn animals come from one rat strain over a limited range of
inhalation concentrations and there are no epidemiologic studies of children exposed to this
compound, the animal results indicate a potential for an increased susceptibility to tumors if
children are exposed. Another limitation is that individual animal data are not available to
determine whether animals with angiosarcomas are more likely to have hepatomas. Without these
data, an assumption of independence was made when combining unit risks across multiple tumor
sites.
The conversion used in this assessment to obtain the human continuous exposure
concentrations in ppm from the corresponding human dose metric in mg/L was a linear one. This
conversion methods seems simplistic given the complexity of the human body. This conversion
may be not be unreasonable, however, because this compound is rapidly and efficiently absorbed,
converted to water-soluble metabolites, and excreted.
h. Application to less-than-lifetime exposure scenarios
Two observations about the early-life studies have implications for how this assessment
would be applied to less-than-lifetime exposure scenarios, particularly during childhood.
1. The exposure period in the early-life experiment (weeks 1-5) does not overlap that of
the chronic experiments (weeks 14-65) used to estimate the inhalation unit risk for
chronic adult exposure. Therefore, the full lifetime cancer risk can be approximated by
adding risks from these nonoverlapping exposure periods.
2. Because the effects of early-life exposure are different from effects of later exposures, it
would not be appropriate to prorate childhood exposures as if they were received at a
proportionately lower rate over a full lifetime.
These observations imply that the potential for increased sensitivity to childhood exposure
is not reflected in the unit risk estimated from later-life exposures. The following examples
illustrate how to combine early-life and later-life unit risk estimates.
Example 1. Full lifetime exposure (birth through death) to 1 ug/m3
The total risk is made up of two components, an early-life risk and a later-life risk.
Risk from early-life exposure: (2.3xl0"6 per ug/m3) x (1 ug/m3) = 2.3xl0"6
Risk from later-life exposure: (2xl0"6 per ug/m3) x (1 ug/m3) = 2xl0"6
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Total risk: 4.3xl0"6
Example 2. Exposure to 2 ug/m3 from ages 30-60
Because exposure begins at age 30, there is no early-life component. The later-life
component is prorated as a duration of 30 years over an assumed lifespan of 70 years.
Risk from early-life exposure: Not applicable
Risk from later-life exposure: (2xl0"6 per ug/m3) x (2 ug/m3) x (30/70) = 1.7xl0"6
Total risk: 1.7xl0"6
Example 3. Exposure to 5 ug/m3 from ages 0-10
Here there is an early-life component that is not prorated. The later-life component is,
however, prorated as 10 out of 70 years.
Risk from early-life exposure: (2.3xl0"6 per ug/m3) x (5 ug/m3) = 1.2xl0"5
Risk from later-life exposure: (2xl0"6 per ug/m3) x (5 ug/m3) x (10/70) = 1.4xl0"6
Total risk: 1.3xl0"5
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Table F-l. Comparison of tumor incidences in male and female Sprague-Dawley rats from 100-hr inhalation
exposures to newborn and mature rats
Angiosarcomas and angiomas (all sites)
Liver hepatomas
Inhaled concentration
(ppm)
Control1
6000 ppm
10,000 ppm
Control1
6000 ppm
10,000 ppm
4 hr/d, 5 d/wk, 5 wk,
starting at age 13 wk
1/277
3/120
2/118
0/277
0/120
1/118
1 hr/d, 4 d/wk, 25 wk,
starting at age 13 wk
1/277
5/118
4/119
0/277
0/118
0/119
4 hr/d, 1 d/wk, 25 wk,
starting at age 13 wk
1/277
4/120
4/119
0/277
2/120
0/119
4 hr/d, 5 d/wk, 5 wk,
starting at age 1 day
1/277
20/42
18/44
0/277
20/42
20/44
One control group served for all exposure patterns
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Table F-2. Comparison of tumor incidences in male and female Sprague-Dawley rats from 5-wk newborn
exposure and 52-wk later-life exposure
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Angiosarcomas and angiomas (all sites)
Liver hepatomas
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5
Inhaled concentration
(ppm)
Control
6000 ppm
10,000 ppm
Control
6000 ppm
10,000 ppm
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7
4 hr/d, 5 d/wk, 52 wk,
starting at age 13 wk
2/58
22/59
13/60
0/58
1/59
1/60
8
9
4 hr/d, 5 d/wk, 5 wk,
starting at age 1 day1'
1/277
20/42
18/44
0/277
20/42
20/44
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aRepeated from table F-l
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Table F-3. Results of PBPK modeling
Inhaled concentration (ppm)
Control
6000 ppm
10,000 ppm
Internal dose of metabolite (mg metabolite / L liver)
0
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404
Equivalent continuous human inhaled concentration (ppm)
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APPENDIX G. RESPONSE TO COMMENTS ON
OTHER SCIENCE ISSUES
1 [To Be Developed]
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